EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS A Critical Role for Sox9 in Notch-Induced Astrogliogenesis and Stem Cell Maintenance SIMONE MARTINI,a KRISTINA BERNOTH,a HEATHER MAIN,b GERMAN DARIO CAMARGO ORTEGA,b URBAN LENDAHL,b URSULA JUST,a RALF SCHWANBECKa Department of Biochemistry, Medical Faculty, Christian-Albrechts-University Kiel, Kiel, Germany; bDepartment of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden a
Key Words. Notch • Neural differentiation • Embryonic stem cells • Transcription factors • Neural stem cell
ABSTRACT Notch signaling is a key regulator of cell-fate decisions and is essential for proper neuroectodermal development. There, it favors the formation of ectoderm, promotes maintenance of neural stem cells, inhibits differentiation into neurons, and commits neural progenitors to a glial fate. In this report, we explore downstream effects of Notch important for astroglial differentiation. Transient activation of Notch1 during early stages of neuroectodermal differentiation of embryonic stem cells resulted in an increase of neural stem cells, a reduction in neurons, an induction of astroglial cell differentiation, and an induction of neural
crest (NC) development. Transient or continuous activation of Notch1 during neuroectodermal differentiation led to upregulation of Sox9 expression. Knockdown of the Notch1-induced Sox9 expression reversed Notch1-induced astroglial cell differentiation, increase in neural stem cells, and the decrease in neurons, whereas the Notch1 effects on NC development were hardly affected by knockdown of Sox9 expression. These findings reveal a critical role for Notch-mediated upregulation of Sox9 in a select set of neural lineage determination steps controlled by Notch. STEM CELLS 2013;31:741–751
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION During neural differentiation, neuroepithelial cells originating from the neural tube differentiate to neurons or glial cells, that is, oligodendrocytes and astrocytes [1]. Neurons differentiate first, followed by differentiation of glial cells. How the relatively simple initial neuroepithelium can give rise to the correct numbers and types of the different subclasses of neurons and glial cells is still rather incompletely understood, but it is becoming increasingly evident that several key intracellular signaling mechanisms play important roles in this process. One signaling mechanism that exerts profound and multifaceted effects on neural differentiation is the Notch signaling pathway [2]. The Notch signaling pathway is a highly conserved mechanism of intercellular communication that controls developmental processes [3]. In mammals, one of the five ligands (Delta 1, 3, 4, Jagged1, and 2) binds to one of the four Notch receptors (Notch1–4) on the surface of neighboring cells. After ligand binding, the Notch intracellular domain (NICD) is proteolytically cleaved from the transmembrane region allowing it to translocate into the nucleus.
Nuclear NICD binds to RBP-J (RBPJk/CBF1/RBPSUH/SUH/ CSL) and assembles into a transcriptional activation complex with coactivator proteins and chromatin remodeling enzymes, finally inducing transcription of downstream target genes [3]. Notch signaling plays important roles at a number of distinct steps during development and maturation of the nervous system, ranging from proliferation within the neuroepithelium to synaptic plasticity in mature neurons [4]. During development of the nervous system, Notch signaling is required for the generation and maintenance of neural stem cells and proper control of neurogenesis in the embryonic and adult brain [5, 6]. Notch signaling is required for gliogenesis in the developing peripheral and central nervous system (CNS), as judged by loss of glial specification when the RBP-J gene was conditionally ablated in the developing nervous system [7]. Furthermore, introduction of NICD into adult neural stem cells promotes glial differentiation [8]. Notch functions are not restricted to the CNS. During neural crest (NC) development, Notch activity first helps to establish the NC domain within the ectoderm via lateral induction and subsequently influences the diversification of NC progeny via lateral inhibition [9]. It is becoming increasingly clear that the Notch
Author contributions: S.M.: collection and assembly of data, data analysis and interpretation, and final approval of manuscript; K.B., H.M., and G.D.C.O.: collection and/or assembly of data and final approval of manuscript; U.L.: data analysis and interpretation, manuscript writing, financial support, and final approval of manuscript; U.J.: concept and design, data analysis and interpretation, provision of study materials, manuscript writing, financial support, and final approval of manuscript; R.S.: Concept and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript. Correspondence: Ursula Just, M.D., Department of Biochemistry, Christian-Albrechts-University of Kiel, Olshausenstr. 40, 24098 Kiel, Germany. Telephone: þ49-431-880-2513; Fax: þ49-431-880-2609; e-mail:
[email protected]; or Ralf Schwanbeck, Ph.D., Department of Biochemistry, Christian-Albrechts-University of Kiel, Olshausenstr. 40, 24098 Kiel, Germany. Telephone: þ49-431-880-2513; Fax: þ49-431-880-2609; e-mail:
[email protected] Received April 3, 2012; accepted for publicaC AlphaMed Press 1066-5099/2013/$30.00/0 tion November 21, 2012; first published online in STEM CELLS EXPRESS January 10, 2013. V doi: 10.1002/stem.1320
STEM CELLS 2013;31:741–751 www.StemCells.com
Notch Induces Astrogliogenesis Via Sox9
742
downstream signaling response is quite cell context dependent [3, 10–12], but how this downstream diversity relates to the fact that Notch signaling at an early stage of neural differentiation blocks differentiation and maintains the stem cell state while at later stages promoting gliogenesis is not established. While hairy and enhancer of split (HES) proteins are induced by Notch signaling and inhibit neural commitment by antagonizing the activity of proneural genes [6, 13], they do not account for the inductive roles of Notch activity during later stages of neuroectodermal development. Sox9 is a transcription factor that plays an important role in the development of the CNS and NC. Sox9 belongs, together with Sox8 and Sox10, to the SoxE group of sex-determining region-related HMG-box family. In the CNS, Sox9 is essential for gliogenesis [14, 15] and necessary and sufficient to initiate the induction of embryonic and adult neural stem cells [16]. It is further expressed directly after specification in NC development [17] and favors NC at the expense of CNS development [18]. As in the CNS, Sox9 blocks differentiation into neurons and supports glial development in the peripheral nervous system [18]. Sox9 has been implicated in Notchmediated gliogenesis as Sox9 expression is downregulated upon RBP-J ablation in the developing nervous system [7]. Due to the fact that Sox9 expression is upregulated following activation of Notch in embryonic stem cells (ESCs) undergoing neural differentiation [10–12], regulation of Sox9 by Notch in the nervous system warrants further analysis. In this study, we examined the role of Sox9 in Notch1mediated control of neural differentiation. Temporally controlled activation of Notch1 in ESC during in vitro differentiation toward the neural lineage revealed roles for Notch signaling in increasing the number of neural stem cells, reducing the number of neurons, inducing glial cell differentiation and inducing NC development. Notch1 upregulated Sox9 expression at all these steps; however, knockdown of the Notch1induced Sox9 expression by siRNA specifically reversed glial and dose-dependently neuronal differentiation as well as stem cell promotion to wild-type levels while having no effect on Notch1-induced NC development. These data define Sox9 as an important Notch downstream gene in controlling a subset of Notch-mediated responses during neuroectodermal development.
MATERIALS
AND
METHODS
Cell Culture and Differentiation The ESC line EB5-NERT [19] expressing a 4-hydroxy-tamoxifen (OHT) inducible form of the Notch1 intracellular domain was grown in ES maintenance medium consisting of KO-Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Karlsruhe, Germany, http://www.lifetechnologies.com) supplemented with 10% knockout serum replacement (Invitrogen, Karlsruhe, Germany, http://www.lifetechnologies.com), 1% pretested fetal calf serum (FCS) (Cambrex/Lonza, Cologne, Germany, http://www.lonza.com/), nonessential amino acids (Invitrogen, Karlsruhe, Germany, http://www.lifetechnologies.com, stock solution diluted 1:100), 2 mM L-glutamine (Invitrogen, Karlsruhe, Germany, http://www.lifetechnologies.com), 5 105 M 2-mercaptoethanol (SigmaAldrich, Munich, Germany, http://www.sigmaaldrich.com), and 10 ng/ml (103 U/ml) cell culture tested leukemia inhibitory factor as described previously [20]. We have shown recently that in vitro differentiation of control EB5 cells was not affected by OHT application [10]. Neural differentiation of EB5-NERT cells was carried out according to the protocol of Ying et al. using a monolayer cell culture in a N2B27 supplemented medium [21]. Briefly,
trypsinized cells were washed, centrifuged, and resuspended in N2B27 medium (49% Neurobasal medium, 49% DMEM/F12 þ Glutamax, 1% B27 supplement, 0.5% N2 supplement, 0.5% L-glutamine, 12.5 lg/ll insulin [all Invitrogen, Karlsruhe, Germany, http://www.lifetechnologies.com], 37.5 lg/ml bovine serum albumin [Sigma-Aldrich, Munich, Germany, http://www.sigmaaldrich.com], and 0.1 mM 2-mercaptoethanol) at a cell density adjusted to 0.125–1 105 cells per well of a six-well plate (Corning, Amsterdam, The Netherlands, http://www.corning.com) in 2 ml medium. For the experiments comprising 1–16 days of differentiation, the medium was changed every 1 or 2 days. Activated Notch1 was induced by addition of 5 or 50 nM OHT for 0–2 days of differentiation or the whole differentiation period as indicated. All differentiation experiments were repeated at least three times. To confirm Sox9 and Pax6 as direct target genes of Notch1 in neuroectodermal differentiation, blocking of protein synthesis was achieved by addition of 50 lg/ml cycloheximide (CHX) for 4 hours. The ESC line Notch1DETetOn was grown and differentiated as described previously [11, 22]. Activated Notch1 was produced in Notch1DETetOn cells by addition of 0.5 lg/ml doxycycline for 0–2 days of differentiation.
Gene Expression Analysis by RT-qPCR RNA was prepared using the RNeasy Plus Kit according to manufacturer’s protocol (Qiagen, Hilden, Germany, http://www.qiagen.com), integrity checked on an agarose gel, and concentrations determined photometrically using the NanoDrop ND1000 (Thermo Scientific, Wilmington, DE, http://www.thermoscientific.com). Total RNA (0.5 or 1 lg) was reversely transcribed with the ‘‘First Strand cDNA Synthesis Kit" (Fermentas/Thermo Scientific, St. Leon-Rot, Germany, http://www.thermoscientific.com) using an oligo(dT)18 primer at 37 C for 60 minutes according to manufacturer’s protocol. In the case of Notch1DETetOn cells, 1 lg total RNA was reversely transcribed with Superscript III Reverse Transcriptase (Invitrogen, Karlsruhe, Germany, http://www.lifetechnologies.com) using an oligo(dT)18 primer at 50 C for 60 minutes according to manufacturer’s protocol. mRNA expression levels of Fgf5, Gapdh, Id3, Nestin, Pax6, Oct4, and Sox9 were measured by qPCR using the TaqMan Assays-on-Demand system (Applied Biosystems/Life Technologies, Darmstadt, Germany, http://www.lifetechnologies.com, Supporting Information Table S1 for Assay number and amplicon sizes). The expression of the genes Brachyury(T), glial fibrillary acidic protein (GFAP), Hprt, Foxd3, Snai1, Snai2, Sox2, Sox17, and Tubb3 was measured by SYBR green detection and S100b by Universal Probe Library (Roche Diagnostics, Rotkreuz, Switzerland, http://www.rocheapplied-science.com) detection (Supporting Information Table S2). All reactions were measured on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Darmstadt, Germany, http:// www.lifetechnologies.com) or on a LightCycler 480 (Roche Diagnostics, Rotkreuz, Switzerland, http://www.roche-applied-science.com) in 384-well PCR-plates in a 10 ll reaction volume using 0.5 ll cDNA. For TaqMan detection, the ‘‘TaqMan 2 Universal Master Mix (No AmpErase UNG)" (Applied Biosystems, Darmstadt, Germany, http://www.lifetechnologies.com) was used, whereas SYBR green detection was performed with ‘‘Power SYBR green Master Mix" (Applied Biosystems, Darmstadt, Germany, http://www.lifetechnologies.com). All assays were tested in initial experiments for amplification efficiency and these efficiencies were used for later calculation of gene expression relative to Gapdh or Hprt [10]. Induction by activated Notch1 was calculated by the formula: I ¼ Expressioninduced/Expressionuninduced.
Flow Cytometry Analysis Expression of different marker proteins was monitored by intracellular flow cytometry analysis using the ‘‘IntraStain Kit" (Dako, Glostrup, Denmark, http://www.dako.com) using antibodies against Nestin, Sox2, Tubb3, and GFAP according to the manual of the manufacturer with minor changes. Briefly, 50 ll IntraStain Reagent A (Fixative) was added to up to 106 cells in 50 ll
Martini, Bernoth, Main et al.
743
medium and incubated for 15 minutes at RT. Cells were washed by addition of 2 ml flow cytometry wash buffer (5% FCS and 0.1% sodium azide in phosphate buffered saline), centrifuged, and the supernatant was discarded. Remaining cells were resuspended in about 10 ll buffer and blocked for 20 minutes by addition of 50 ll IntraStain Reagent B þ0.1% NP40 þ 3% normal goat serum. Subsequently, the respective primary antibody was added and incubated for 20 minutes at 4 C. Since antibodies for the GFAP and Pax6 proteins were not directly labeled, the protocol was extended for an additional washing step with 2 ml flow cytometry wash buffer and staining with a secondary antibody in 50 ll IntraStain Reagent B þ0.1% NP40 for 20 minutes at 4 C in the dark, and washed again. Used antibodies and isotype controls are itemized in Supporting Information Table S3. Filtered samples were analyzed on a FACSCanto (Becton Dickinson, Heidelberg, Germany, http://www.bd.com) using the FACS Diva software (Version 5.03).
Knockdown of Sox9 and Pax6 EB5 NERT or Notch1DETetOn cells were trypsinized, centrifuged, counted, and then resuspended and seeded in ES maintenance medium into six-well plates (Corning, Amsterdam, The Netherlands, http://www.corning.com). After 6–8 hours, the medium was changed to N2B27, and Notch1 induction as well as the transfection for knockdown was started. Two micromolar (Pax6) or 4 lM (Sox9) siRNA stock solutions of Sox9 or Pax6 pool of siRNAs (both Dharmacon/Thermo Scientific, Wilmington, DE, http://www.thermoscientific.com) were mixed with N2B27-medium in a 200 ll volume and incubated for 5 minutes at RT. In parallel, 4 ll DharmaFECT1 (Dharmacon/Thermo Scientific, Wilmington, DE, http://www.thermoscientific.com) was mixed with 196 ll serum-free medium and also incubated for 5 minutes at RT, and then the diluted siRNA was added to this mix and incubated for an additional 20 minutes at RT. This transfection mix was added to the cells in 2 ml N2B27 medium in the six-well plates. The final concentration of Sox9 and Pax6 siRNA was 100 nM and 50 nM, respectively. As a control, a 100 nM pool of nontargeting siRNA (negative control siRNA, Dharmacon/Thermo Scientific, Wilmington, DE, http://www.thermoscientific.com) or transfection reagent only was used.
Data Analysis and Statistics Statistics were analyzed as paired data for the Student’s t test or, in the case of uneven sample numbers, as heteroscedastic data. The qPCR data were calculated by comparison of the DCt values of the different sample populations, whereas flow cytometric results were calculated on basis of the resulted induction values. Different levels of significance according to the p values are indicated by *, p < .05; **, p < .01; and ***, p < .005.
Figure 1. Activated Notch1 induces morphological changes during neuroectodermal differentiation. EB5 NERT cells were differentiated up to 16 days in neuroectodermal conditions in the presence or absence of 50 nM OHT. Wild-type differentiations spontaneously formed neural rosettes within 6 days (A) and by 16 days extensive neuronal networks were observed (C), which stained positive for TuJ1(Tubb3) (E—green). In the presence of constant OHT treatment, neural clusters surrounded by large dispersed cells appeared at day 5 (B) and by day 16 an increase in bipolar cells was seen (D), correlating with a decrease in TuJ1-positive neuronal axons (F—green). While few GFAP-positive cells were observed after 16 days of wildtype differentiation (G—orange), many GFAP-positive cells were observed with constant OHT (H—orange). 4’,6-diamidino-2-phenylindole (DAPI) staining is shown in blue. All images are with a 10 objective. Abbreviations: GFAP, glial fibrillary acidic protein; OHT, 4-hydroxy-tamoxifen.
RESULTS Notch1 Signaling During Neuroectodermal Commitment Promotes Neural Stem Cells, Inhibits Neuronal Differentiation, and Induces Glial and NC Differentiation To analyze the role of Notch1 signaling during neuroectodermal differentiation of ESCs in vitro, we took advantage of a previously established ESC system, EB5-NERT, in which Notch1 signaling can be activated by OHT [19, 23]. EB5NERT cells were differentiated in a monolayer culture favoring the generation of neuroectodermal cells. Notch1 signaling was either continuously activated by the addition of OHT throughout culturing or transient during early stages of differentiation (0–2 days). In the absence of OHT, the EB5-NERT cells differentiated into neural stem cells forming rosettes by days 4–6 of neuroectodermal differentiation and further from www.StemCells.com
day 8 onward into mature neuronal cells expressing the neuronal marker protein Tubb3 and from day 10 onward into glial cells expressing the glial cell marker protein GFAP (Fig. 1A, 1C, 1E, 1G). Continuous as well as transient activation of Notch1 by the application of OHT profoundly altered differentiation (Fig. 1B, 1D, 1F, 1H): by day 5, large dispersed cells with a flattened morphology appeared surrounding neuronal cluster-like colonies. These cells then further differentiated into small rosettes consisting of bipolar cells with small processes resembling neural stem cells. In the presence of OHT, only few bipolar or multipolar neuronal precursor cells expressing Tubb3 were obtained. In contrast, a considerably higher number of GFAP-positive glial cells were formed in the presence of OHT. To further characterize the alterations in neuroectodermal differentiation induced by activated Notch1, we compared
Notch Induces Astrogliogenesis Via Sox9
744
Figure 2. Upregulation of Sox9 expression by activated Notch1 correlates with upregulation of neural stem cell marker Nestin and later during differentiation also with glial marker GFAP und astrocytic marker S100b during in vitro neuroectodermal differentiation. EB5 NERT cells were differentiated up to 16 days and were treated for the whole period (continuous OHT, black lines or bars) or for 2 days with 50 nM OHT (0–2 days OHT, dark gray lines or bars) or left untreated (OHT, light gray lines). The expression of genes was measured via RT-qPCR and normalized to Gapdh or Hprt. Time kinetics was sometimes shifted, resulting in high SD values. Thus, results shown in A, C, E, G are representative examples of duplicate measurements. Experiments were repeated four to seven times (OHT and continuous OHT) or three to five times (0–2 days OHT) with similar results. (B, D, F, H): Induction values expressed as induction by activated Notch1 compared to not-induced samples were calculated from expression values from A, C, E, G. Mean values of four to seven experiments (continuous OHT) or three to five experiments (0–2 days OHT) are shown as well as the calculated SDs. Student’s t test revealed statistical significant changes induced by OHT (*, p < .05; **, p < .01; ***, p < .005). Abbreviations: GFAP, glial fibrillary acidic protein; OHT, 4-hydroxy-tamoxifen.
RNA expression levels of Sox9, (Fig. 2) as well as Oct4, Sox2, Snai1, and Snai2 (inductions by Fig. 3 and normalized expression
Nestin, GFAP, and S100b Tubb3, Pax6, Id3, Foxd3, activated Notch1 shown in levels shown in Supporting
Information Fig. S1) throughout neural differentiation in the presence of OHT, either continuously or only for the first 2 days of differentiation, or in absence of OHT. Continuous as well as transient Notch1 signaling induced expression of
Martini, Bernoth, Main et al.
745
Figure 3. Notch1 activation upregulates expression of markers for neural stem cells and NC and downregulates neuronal marker Tubb3 during in vitro neuroectodermal differentiation. EB5 NERT cells were differentiated up to 16 days and were treated for the whole period (black bars) or with a 2-day (days 0–2) treatment with 50 nM OHT (gray bars). The expression of marker genes Oct4 (A), Sox2 (B), Pax6 (C), Tubb3 (D), Id3 (E), Foxd3 (F), Snai1 (G), and Snai2 (H) was measured via RT-qPCR and expressed as induction by activated Notch1 compared to not-induced samples. Shown are mean values of four to seven experiments (continuous OHT) or three to five experiments (0–2 days OHT) and the calculated SDs. Student’s t test revealed statistical significant changes induced by OHT (*, p < .05; **, p < .01; ***, p < .005). Gray fields indicate specificity of marker genes. Normalized expression values are shown in Supporting Information Figure S1. Abbreviations: ESC, embryonic stem cell; NC, neural crest; NSC, neural stem cells; OHT, 4-hydroxy-tamoxifen.
Sox9, a key transcription factor of neural stem cells, glial precursors, and NC cells [17]. Activation of Notch1 during the first 2 days was sufficient to induce an upregulation of Sox9 that lasted also after Notch1 was downregulated (Fig. 2A, www.StemCells.com
2B). A similar upregulation of expression was seen also for the stem cell marker Nestin (Fig. 2C, 2D). The glial cell marker GFAP and the astrocytic marker S100b were upregulated by Notch1 signaling at late stages of differentiation
746
consistent with an induction of glial progenitor cells and mature astrocytes by activated Notch1 (Fig. 2E, 2G, 2F, 2H). Interestingly, transient Notch1 activation favored the induction of GFAP expression, whereas continuous activation of Notch1 results in an enhanced S100b expression. Continuous as well as transient activation of Notch1 signaling during neuroectodermal induction resulted in decreased levels of the ESC marker Oct4 (Fig. 3A and Supporting Information Fig. S1A), a transient decrease of embryonic and neural stem cell marker Sox2 during the first 2 days of neuroectodermal differentiation with a later increase in Sox2, indicating neural differentiation was increased by OHT from day 10 onward (Fig. 3B and Supporting Information Fig. S1B). OHT treatments induced a transient increase of the neural and neural stem cell marker Pax6 during the first 2 days of differentiation with a subsequent decrease thereafter and for transient Notch1 activation a delayed increase from day 12 onward (Fig. 3C and Supporting Information Fig. S1C). The neuronal marker Tubb3 was strongly decreased from day 8 onward after continuous as well as transient activation of Notch1 signaling (Fig. 3D and Supporting Information Fig. S1D). Continuous as well as temporal activation of Notch1 during neuroectodermal induction upregulated expression of Id3 (Fig. 3E and Supporting Information Fig. S1E), which is involved in selfrenewal and proliferation of neural stem and NC progenitor cells [24]. Id3 expression remained high in presence of continuous Notch1 activation but decreased from day 12 onward after transient activation of Notch1 signaling, indicating that the cells resume differentiation upon termination of Notch1 activation (Fig. 3E and Supporting Information Fig. S1E). We next assessed the expression of genes involved in NC specification, that is, Foxd3, Snai1, and Snai2. Activation of Notch1 increased expression of Foxd3, Snai1, and Snai2 from day 4 onward, with a maximum around days 6–8 of differentiation when Notch1 signaling was transiently activated (Fig. 3F–3H and Supporting Information Fig. S1F–S1H), suggesting a strong induction of NC by Notch1 signaling. We further analyzed the expression of markers representative of other lineages, that is, the epiblast and primitive ectoderm marker Fgf5, the mesoderm marker Brachyury(T), and the endoderm marker Sox17. In line with differentiation into primitive ectoderm, Fgf5 expression was upregulated in differentiating cultures between day 2 and 4 without activation of Notch1 signaling, while continuous as well as temporal activation of Notch1 signaling strongly downregulated the expression of Fgf5 (Supporting Information Fig. S2A). Downregulation of Fgf5 correlated with an early upregulation of the neural stem cell markers Pax6, Sox9, and Nestin, indicative of an accelerated differentiation into the neuroectoderm. Irrespective of Notch1 activation, the neuroectodermally differentiating cultures contained hardly any endodermal or mesodermal cells, as indicated by the absence of Sox17 expression and the low levels of Brachyury(T) in presence or absence of OHT (Supporting Information Fig. S2B). Since Notch1 signaling may influence cell death, we next determined the number of apoptotic cells in our differentiating cultures in the presence or absence of activated Notch1. Irrespective of transient or continuous activation of Notch1 signaling, the number of apoptotic cells was generally less than 5% throughout the cultures up to 15 days (Supporting Information Fig. S3). Thus, cell death is not playing a role for the outcome of Notch induced cell fate decisions in our neural differentiating cultures. To further quantify the observed changes in neuroectodermal differentiation, the developing cell populations were characterized by flow cytometry at days 12–14 of neuroectodermal differentiation in presence or absence of Notch1 signaling using expression of Tubb3 for neuronal precursor cells, of
Notch Induces Astrogliogenesis Via Sox9
GFAP for glial precursor cells and astrocytes, of Nestin for neural precursor cells, and of Sox2 plus Nestin for neural stem cells. As shown in Figure 4A, 4B, continuous activation of Notch1 increased the number of stem cells, neural precursor cells, and glial cells, whereas it decreased the number of neuronal precursor cells. Similarly, transient activation of Notch1 during the first 2 days of neuroectodermal differentiation increased the number of stem cells, neural precursors, and glial cells while decreasing the number of neuronal precursors (Fig. 4C, 4D). The number of stem cells however was increased to a much higher extent when Notch1 signaling was constantly active, suggesting that Notch signaling keeps the cells temporarily in an undifferentiated state but alters lineage decisions permanently (Fig. 4). To corroborate the data in an alternative ESC line, where Notch activation can be temporally controlled, we used the Notch1DETetOn ESC line, in which Notch1DE is induced by doxycycline via the TetOn system [11, 22]. Remarkably similar effects were found after induction of Notch signaling by Doxycyclin: Sox9 expression is induced early during differentiation, correlating then with expression of the stem cell marker Nestin, and is further induced later during differentiation, correlating then with the expression of glial marker GFAP and inversely with expression of neuronal marker Tubb3 (Supporting Information Fig. S4).
The Notch1-Mediated Lineage Decision Between Neuronal and Glial Differentiation Is Mediated by Sox9 To explore whether Sox9 expression was directly regulated by Notch1, we transiently activated Notch1 by addition of OHT for 4 hours in the presence of the protein synthesis inhibitor CHX. Induction of Notch1 in neuroectodermally differentiating cells resulted in an increase of Sox9 expression despite the presence of CHX (Supporting Information Fig. S5), indicating a transcriptional regulation. Next, we sought to dissect the role of Notch1-induced upregulation of Sox9 expression for neuroectodermal commitment of ESCs. To this end, we used a siRNA-mediated Sox9 knockdown approach to specifically decrease Notch1-induced Sox9 levels back to control levels. The Sox9 siRNA knockdown efficiently decreased the Sox9 RNA level and resulted in a Sox9 protein level comparable to the control (Fig. 5A and Supporting Information Fig. S6A–S6C). Reducing Sox9 expression levels down to control levels during Notch1 activation during the first 5 days of neuroectodermal differentiation had a strong influence on the Notch1-induced lineage decision between glial cells, in particular astrocytes, and neuronal cells as analyzed by GFAP, S100b, and Tubb3 expression. Notch1 activation, at normal Sox9 levels, increased the number of GFAP-positive cells, S100b-positive cells, and GFAP/S100b double-positive cells and decreased the number of Tubb3-positive neuronal cells (Fig. 5B–5F). While GFAPpositive cells were increased with transient and continuous as well as high (50 nM OHT) and low (5 nM OHT) induction of Notch1 signaling, the suppression of Tubb3-positive cells required a stronger Notch1 activation by either a higher level temporarily (50 nM OHT) or by applying OHT continuously. Knockdown of Sox9 expression reversed the Notch1induced increase in GFAP-positive glial cells and GFAP/ S100b-positive astrocytes back to the levels seen without Notch1 induction (Fig. 5B–5F). While the suppression of Tubb3-positive cells by transient Notch1 signaling could be reverted with Sox9 knockdown (Fig. 5D), continuous Notch1 signaling inhibited Tubb3 induction in the presence and absence of Notch1-induced Sox9 induction. This suggests that
Martini, Bernoth, Main et al.
747
Figure 4. Activated Notch1 induces differentiation into glia and neural stem cells and inhibits the generation of neuronal precursor cells. EB5 NERT cells were grown for 12–14 days along the neuroectodermal lineages and analyzed by flow cytometry for different marker proteins. Cells were induced continuously (A, B) or from day 0 to 2 (C, D) with 50 nM OHT. The resulted fold change by OHT application was calculated in (B) and (D). An increase in NestinþSox2-positive cells, an increase in GFAP-positive cells and decrease in Tubb3 (TuJ1)-positive cells were seen with continuous (A) or day 0–2 (C) OHT treatment. Student’s t test revealed statistical significant changes by OHT (*, p < .05; **, p < .01; ***, p < .005). Results shown are from three to six (continuous OHT) or three to four independent experiments (0–2 days OHT) including the respective SDs. Abbreviations: GFAP, glial fibrillary acidic protein; OHT, 4-hydroxy-tamoxifen.
Notch effects independent of early Sox9 induction also contribute to neuronal suppression, possibly by Notch-Hes inhibition of neuronal differentiation. To further corroborate the significance of Sox9 for the Notch-mediated lineage decision between glial and neuronal cells, we knocked down Sox9 during neuroectodermal differentiation of Notch1DETetOn cells. Activation of Notch1 signaling by a low-dose of Dox upregulated Sox9 expression early (day 1) and late (between day 5 and 8) during neuroectodermal differentiation (Supporting Information Figs. S4, S7). These two upregulations of Sox9 expression corresponded well with the inductions seen with the OHT-inducible system in EB5-NERT cells (Fig. 2). While in both systems Notch1 induced Sox9 expression during early and late neuroectodermal differentiation, differences were seen in the kinetics of differentiation. EB5-NERT cells differentiated slower that the Ainv15-Notch1DETetOn cells (compare Supporting Information Fig. S4 with Fig. 2). Despite the different kinetics, knockdown of Notch1-induced Sox9 expression (Supporting Information Fig. S7) efficiently reverted Notch1-induced glial differentiation also in the Tet-inducible Ainv15 ESC (Fig. 5G). In line with our results with NERT cells (Fig. 5E), a low level of Notch1 activation by Dox in the Notch1DETetOn cells was not sufficient to decrease the number of Tubb3-positive cells by day 14, although Tubb3 expression was significantly www.StemCells.com
downregulated by activated Notch1. These results further underline the importance of dose and timing of Notch signaling for lineage decision events. Surprisingly, only when Notch1 signaling was activated by Dox in the Notch1DETetOn cells, the induction of neural stem cells by activated Notch1 could be counteracted by reducing Sox9 levels with siRNA (Fig. 5B–5E, 5G). In the Notch1DETetOn cells, the treatment with Sox9 siRNA decreased the early and late induction of Sox9 by activated Notch1 (Supporting Information Fig. S7). The duration of Sox9 induction in response to 2 days of Notch1 activation differed between the EB5-NERT and the Notch1DETetOn cells: in EB5-NERT cells, Sox9 was increased for 12 days, whereas it was increased for 6 days in the Notch1DETetOn cells (compare Fig. 2 with Supporting Information Fig. S4). When Notch1 signaling was switched on continuously or transiently during neural differentiation by OHT in the NERT system, the number of neural stem cells remained at a high level, despite downregulation of the early Notch1-induced Sox9 expression. It is likely that the remaining Sox9 induction at later time points (Fig. 2) prevented stem cell differentiation in EB5 NERT cells treated with OHT and Sox9 siRNA. Taken together, our results further emphasize that the intensity and duration of the Notch-induced Sox9 upregulation are critical for Notch-mediated stem cell arrest.
748
Notch Induces Astrogliogenesis Via Sox9
Figure 5. Knockdown of Sox9 reverts effects of activated Notch1 on neuroectodermally differentiating cells. (A): Sox9 protein was efficiently downregulated by Sox9 siRNA. EB5 NERT cells were treated with 100 nM Sox9 siRNA and Notch1 signaling was immediately induced by 50 nM OHT. After 2 days of neuroectodermal differentiation, proteins were harvested, separated on a SDS-PAGE, and a Western blot was stained for Sox9 expression. Samples were loaded in duplicates of independent experiments. Actin was used as a loading control. In total, the experiment was repeated three times with virtually identical results. (B–F): Reversion of Notch1-induced effects by Sox9 knockdown using the NERT system. EB5 NERT cells were kept as described in Figure 4, but additionally treated with Sox9 siRNA in the initial phase of differentiation. The expression of the proteins Nestin, Sox2, GFAP, and Tubb3/Tuj1 was analyzed by intracellular flow cytometry after 12–14 days of differentiation. (B, C): Continuous OHT treatment. (D–F): Transient Notch1 induction from day 0 to 2. (B, D, F): Fifty nanomolar OHT treatment. (C, E): Five nanomolar OHT treatment. (G): Reversion of Notch1-induced effects by Sox9 knockdown using the TetOn system. Ainv15 embryonic stem cells carrying the Notch1DETetOn system were differentiated for 14 days according to the neuroectodermal differentiation protocol and Notch1 signaling was activated by the addition of Dox from day 0 to 2. Additionally, the cells were treated with Sox9 siRNA in the initial phase of differentiation. The expression of the proteins Nestin, Sox2, GFAP, and Tubb3/Tuj1 was analyzed by intracellular flow cytometry after 12–14 days of differentiation. Results shown are mean values and respective SDs values of three to six independent experiments. Student’s t test revealed statistical significant changes between fractions (*, p < .05; **, p < .01; ***, p < .005). Abbreviations: GFAP, glial fibrillary acidic protein; OHT, 4-hydroxy-tamoxifen.
Martini, Bernoth, Main et al.
749
To analyze whether preventing Notch1-mediated upregulation of Sox9 during early neuroectodermal differentiation of ESC would also affect NC development, we knocked down the Notch1-induced Sox9 expression and analyzed the expression of Snai1, Snai2, and Id3. The upregulation observed by addition of OHT in the EB5-NERT cells from day 0 to 2 was only partially affected by knockdown of Sox9 expression: there was a partial downregulation of Id3, but not of Snai1 and Snai2 (Supporting Information Fig. S8), suggesting that the early Sox9 induction contributes to only some extent to the Notch-induced specification and/or self-renewal of NC stem cells.
Downregulation of Pax6 Expression by siRNA Is Not Sufficient to Revert Notch1-Induced Changes During Neuroectodermal Differentiation In addition to Sox9, we have recently identified Pax6 as a Notch1 target gene in ESC [10]. Pax6 plays a crucial role in neuroectodermal development [25] and we therefore assessed the importance of Pax6 upregulation during neuroectodermal differentiation of ESC. First, we confirmed that activated Notch1 upregulates Pax6 in ESC and during early neuroectodermal differentiation (Fig. 3C and Supporting Information Fig. S1C). Upregulation was restricted to the first 2 days of neuroectodermal differentiation, and during days 4–10 of neural differentiation, activation of Notch1 downregulated Pax6 expression (Fig. 3C and Supporting Information Fig. S1C). Knockdown of Pax6 expression by siRNA, as expected, decreased Notch1-induced upregulation of Pax6 protein and RNA levels (Fig. 6A and Supporting Information Fig. S9). Manipulating Pax6 expression levels back to control levels during Notch1 activation during the first days of neuroectodermal differentiation had no significant influence on the Notch1-induced lineage decision between glial and neuronal cells or on the Notch1-induced increase in neural stem and progenitor cells as analyzed by GFAP, Tubb3, Nestin, and Sox2 expression (Fig. 6), suggesting that Pax6 upregulation is not sufficient to mediate these effects of Notch.
DISCUSSION Notch signaling controls cell fate decisions in many different cellular contexts and often acts at different steps during cell lineage progression. This is indeed the case in neural differentiation, where Notch signaling supports the generation and maintenance of neural stem cells and is required for gliogenesis. In this report, we have addressed how Notch signaling regulates neuroectodermal cell fate decisions, and we provide evidence for a critical role of Sox9 in promotion of neural stem cell maintenance and astroglial differentiation. Using inducible systems to activate Notch1 during in vitro differentiation of ESC along the neuroectodermal lineage, we identified Sox9 as a direct Notch target gene and observed several changes in neuroectodermal development (summarized in Fig. 7). Briefly, our data show that continuous or transient activation of Notch1 during neural differentiation of ESC resulted in an increase in neural stem cells, a block in generation of neurons, and an increase in immature glial cells and astrocytes. This extends previous studies, where ligand-specific Notch activation during embryonic neurogenesis promotes and accelerates differentiation of ESC, supports development of neuroectodermal cells, and inhibits differentiation along mesodermal lineages [19, 26, 27]. Subsequently, Notch signaling is essential for maintenance of neural stem and progenitor cells and blocks differentiation into neurons [6, 28], whereas www.StemCells.com
Figure 6. Pax6 can be downregulated by siRNA but downregulation of Pax6 has no significant effect on Notch1-induced neurogenesis. EB5 NERT cells were differentiated as described in Figure 4 in the presence or absence of Pax6 siRNA and 50 nM OHT treatment. (A): After 24 hours of neuroectodermal differentiation, the cells were analyzed for Pax6-positive cells by intracellular flow cytometry. The results shown are mean values and SDs of three independent experiments. The significant increase of Pax6 protein expression by activated Notch1 induction was reverted significantly by application of Pax6 siRNA (*, p < .05). (B, C): The expression of the proteins Nestin, Sox2, GFAP, and Tubb3/Tuj1 was followed by intracellular flow cytometry after 12–14 days of differentiation to analyze the influence of Pax6 knockdown with (B) continuous or (C) transient OHT treatment (50 nM). Results shown are mean values and SDs of four to six independent experiments. Student’s t test revealed statistical significant changes between fractions (*, p < .05; **, p < .01; ***, p < .005). Abbreviations: GFAP, glial fibrillary acidic protein; OHT, 4hydroxy-tamoxifen.
it induces gliogenic differentiation [5, 7, 29–31]. Interestingly, transient activation of Notch1 during early neuroectodermal ESC differentiation was sufficient to irreversibly switch neurogenesis to gliogenesis, suggesting that Notch functions at this stage as an instructive rather than a permissive signal. In line with this, temporal activation of Notch by its ligand Jagged in neural stem cells for 5 hours is sufficient to irreversibly inhibit differentiation into neurons [32]. Furthermore, an instructive glial determination and a block of neuronal differentiation were observed in NC stem cells after activation of Notch by ligand stimulation for 24 hours [30].
750
Notch Induces Astrogliogenesis Via Sox9
Figure 7. Model of functions of activated Notch on neuroectodermal differentiation and neurogenesis and their mediation by Sox9. Activated Notch initially accelerates neuroectodermal and neural differentiation, contributes to the induction and maintenance of glia and neural stem cells, and promotes the generation of radial glia cells in the peripheral and central nervous system (green arrows). Furthermore, it leads to the inhibition of differentiation into neurons (red blunt arrows). In this work, we show that Sox9 plays an essential role in the mediation of Notch induced astro-gliogenesis as well as for maintenance of neural stem cells. Additionally, Sox9 is involved in the inhibition of neurons. Abbreviation: ES, embryonic stem.
Interestingly, Sox9 played an important role in mediating the Notch response in promotion of astroglial differentiation and neural stem cell maintenance, while it was considerably less important for maintaining the block of neurogenesis and for inducing NC differentiation. The Notch-mediated block of neurogenesis may instead be controlled by the Hes/Hey family of transcription factors that inhibit proneural gene expression [13, 33]. The finding that Sox9 was a critical mediator of Notch1induced glial promotion corroborates a previous report, where selective ablation of Notch in the developing nervous system reduced glial differentiation, and was accompanied by reduced Sox9 expression, although the importance of Sox9 was never directly tested [7]. In the specialized setting of eye development, there is evidence that group E proteins Sox8 and 9 are involved in mediating Notch effects for generation of Mu¨ller glia cells, which are part of the vertebrate retina [34]. However, it was not shown to be a general mechanism for glia cell development and group E Sox proteins were not confirmed as direct target genes of the Notch signaling. Sox9 induced by activated Notch1 is an essential mediator also for chondrogenic lineage determination [35], which lends further support to the model that Notch signaling regulates lineage decisions via direct induction of lineage-determining transcription factors [12]. In accordance with this notion, in Drosophila, Notch signaling is required for expression of the transcription factor gcm, which directs glial specification [36]. Several studies suggest that Notch signaling plays a role in establishing the NC domain within the ectoderm and subsequently in diversifying the fates of cells that arise from NC [9, 37]. In this study, we observed the appearance of a population of large cells with a high migratory potential after activation of Notch1 signaling during neuroectodermal differentiation of ESC and an increase in Snai1, Snai2, and Foxd3 expression from day 4 onward, indicating induction of differentiation into immature NC cells. Promotion of NC lineage progression is in contrast to a previous report, which, using
the same ESC differentiation protocol, did not observe NC or glial cells when Notch1 was constitutively expressed from the ROSA26-locus [26]. The reason for this discrepancy is not clear, but may at least in part reflect different cell densities in the two studies, as cell density is known to influence the effects of ESC differentiation [38]. Alternatively, the constitutive activation of Notch prior to neural differentiation [26] may have led to selection of cells that had lost the capacity to differentiate into NC and glial cells, whereas they retained neuronal differentiation capacity. In conclusion, the data presented here shed new light onto how Notch signaling can execute cell fate decisions at various steps during a cell lineage progression, and the observation that Sox9 is critical for a subset of fate choices during neural differentiation provides new insights into how Notch promotes glial differentiation.
CONCLUSIONS In conclusion, this is the first study to demonstrate that Notch1 signaling induces glial cell differentiation of ESCs and neural stem cell self-renewal via the direct upregulation of the transcription factor Sox9. Furthermore, we show that Sox9 is only partially involved in the Notch1-induced decrease in neurons and does not play a major role for Notch1 effects on NC development. Our results identify Sox9 as a critical and specific mediator of Notch controlled neural lineage commitment.
ACKNOWLEDGMENTS We are very grateful to S. Horn, M. Boss, and K. Macha for their skillful technical assistance, Dr. Michael Wegner for providing the Sox9 antibody, and Dr. Claudia Lange for helpful
Martini, Bernoth, Main et al.
suggestions. This work was supported by funding from the DFG SFB 415 project B8 to U.J., and by funding from the Swedish Research Council (project grant and DBRM), Knut och Alice Wallenbergs Stiftelse (WIRM), Cancerfonden, and EU (NotchIT) to U.L.
REFERENCES 1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19
Rowitch DH, Kriegstein AR. Developmental genetics of vertebrate glial-cell specification. Nature 2010;468:214–222. Pierfelice T, Alberi L, Gaiano N. Notch in the vertebrate nervous system: An old dog with new tricks. Neuron 2011;69:840–855. Andersson ER, Sandberg R, Lendahl U. Notch signaling: Simplicity in design, versatility in function. Development 2011;138:3593–3612. Ables JL, Breunig JJ, Eisch AJ et al. Not(ch) just development: Notch signalling in the adult brain. Nat Rev Neurosci 2011;12:269–283. Tanigaki K, Honjo T. Two opposing roles of RBP-J in Notch signaling. Curr Top Dev Biol 2010;92:231–252. Imayoshi I, Sakamoto M, Yamaguchi M et al. Essential roles of Notch signaling in maintenance of neural stem cells in developing and adult brains. J Neurosci 2010;30:3489–3498. Taylor MK, Yeager K, Morrison SJ. Physiological Notch signaling promotes gliogenesis in the developing peripheral and central nervous systems. Development 2007;134:2435–2447. Tanigaki K, Nogaki F, Takahashi J et al. Notch1 and Notch3 instructively restrict bFGF-responsive multipotent neural progenitor cells to an astroglial fate. Neuron 2001;29:45–55. Cornell RA, Eisen JS. Notch in the pathway: The roles of Notch signaling in neural crest development. Semin Cell Dev Biol 2005;16: 663–672. Meier-Stiegen F, Schwanbeck R, Bernoth K et al. Activated Notch1 target genes during embryonic cell differentiation depend on the cellular context and include lineage determinants and inhibitors. PLoS One 2010;5:e11481. Main H, Lee KL, Yang H et al. Interactions between Notch- and hypoxia-induced transcriptomes in embryonic stem cells. Exp Cell Res 2010;316:1610–1624. Schwanbeck R, Martini S, Bernoth K et al. The Notch signaling pathway: Molecular basis of cell context dependency. Eur J Cell Biol 2011;90:572–581. Kageyama R, Ohtsuka T, Shimojo H et al. Dynamic Notch signaling in neural progenitor cells and a revised view of lateral inhibition. Nat Neurosci 2008;11:1247–1251. Stolt CC, Lommes P, Sock E et al. The Sox9 transcription factor determines glial fate choice in the developing spinal cord. Genes Dev 2003;17:1677–1689. Stolt CC, Wegner M. SoxE function in vertebrate nervous system development. Int J Biochem Cell Biol 2010;42:437–440. Scott CE, Wynn SL, Sesay A et al. SOX9 induces and maintains neural stem cells. Nat Neurosci 2010;13:1181–1189. Hong CS, Saint-Jeannet JP. Sox proteins and neural crest development. Semin Cell Dev Biol 2005;16:694–703. Cheung M, Briscoe J. Neural crest development is regulated by the transcription factor Sox9. Development 2003;130:5681–5693. Schroeder T, Meier-Stiegen F, Schwanbeck R et al. Activated Notch1 alters differentiation of embryonic stem cells into mesodermal cell lineages at multiple stages of development. Mech Dev 2006;123: 570–579.
751
DISCLOSURE
OF
POTENTIAL CONFLICTS INTEREST
OF
The authors indicate no potential conflicts of interest. 20 Schroeder T, Fraser ST, Ogawa M et al. Recombination signal sequence-binding protein Jkappa alters mesodermal cell fate decisions by suppressing cardiomyogenesis. Proc Natl Acad Sci USA 2003;100: 4018–4023. 21 Ying QL, Stavridis M, Griffiths D et al. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 2003;21:183–186. 22 Das D, Lanner F, Main H et al. Notch induces cyclin-D1-dependent proliferation during a specific temporal window of neural differentiation in ES cells. Dev Biol 2010;348:153–166. 23 Schroeder T, Just U. Notch signalling via RBP-J promotes myeloid differentiation. EMBO J 2000;19:2558–2568. 24 Light W, Vernon AE, Lasorella A et al. Xenopus Id3 is required downstream of Myc for the formation of multipotent neural crest progenitor cells. Development 2005;132:1831–1841. 25 Osumi N, Shinohara H, Numayama-Tsuruta K et al. Concise review: Pax6 transcription factor contributes to both embryonic and adult neurogenesis as a multifunctional regulator. Stem Cells 2008;26:1663–1672. 26 Lowell S, Benchoua A, Heavey B et al. Notch promotes neural lineage entry by pluripotent embryonic stem cells. PLoS Biol 2006;4:e121. 27 Ramasamy SK, Lenka N. Notch exhibits ligand bias and maneuvers stage-specific steering of neural differentiation in embryonic stem cells. Mol Cell Biol 2010;30:1946–1957. 28 Shimojo H, Ohtsuka T, Kageyama R. Oscillations in notch signaling regulate maintenance of neural progenitors. Neuron 2008;58:52–64. 29 Piper M, Barry G, Hawkins J et al. NFIA controls telencephalic progenitor cell differentiation through repression of the Notch effector Hes1. J Neurosci 2010;30:9127–9139. 30 Morrison SJ, Perez SE, Qiao Z et al. Transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells. Cell 2000;101:499–510. 31 Robinson AP, Foraker JE, Ylostalo J et al. Human stem/progenitor cells from bone marrow enhance glial differentiation of rat neural stem cells: A role for transforming growth factor beta and Notch signaling. Stem Cells Dev 2011;20:289–300. 32 Grandbarbe L, Bouissac J, Rand M et al. Delta-Notch signaling controls the generation of neurons/glia from neural stem cells in a stepwise process. Development 2003;130:1391–1402. 33 Bertrand N, Castro DS, Guillemot F. Proneural genes and the specification of neural cell types. Nat Rev Neurosci 2002;3:517–530. 34 Muto A, Iida A, Satoh S et al. The group E Sox genes Sox8 and Sox9 are regulated by Notch signaling and are required for Muller glial cell development in mouse retina. Exp Eye Res 2009;89:549–558. 35 Haller R, Schwanbeck R, Martini S et al. Notch1 signaling regulates chondrogenic lineage determination through Sox9 activation. Cell Death Differ 2012;19:461–469. 36 Udolph G, Rath P, Chia W. A requirement for Notch in the genesis of a subset of glial cells in the Drosophila embryonic central nervous system which arise through asymmetric divisions. Development 2001; 128:1457–1466. 37 Mead TJ, Yutzey KE. Notch pathway regulation of neural crest cell development in vivo. Dev Dyn 2012;241:376–389. 38 Ying QL, Smith AG. Defined conditions for neural commitment and differentiation. Methods Enzymol 2003;365:327–341.
See www.StemCells.com for supporting information available online.