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Title: Neural differentiation is increased by GSK-3β inhibition and decreased by
tankyrase inhibition in human neural precursor cells
Running head: Wnt signaling in human neural precursors
Authors: Michael Telias1,†, Dalit Ben-Yosef1,*.
Affiliations: 1Wolfe PGD-SC Lab, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv
Sourasky Medical Center & Department of Cell and Developmental Biology Sackler
Medical School, Tel-Aviv University.
*Corresponding author: Dalit Ben-Yosef, dalitb@tlvmc.gov.il
†Current Affiliation: Department of Molecular and Cell Biology, University of California
Berkeley, California, USA.
Abstract:
Glycogen synthase kinase-3β (GSK-3β) and tankyrase-1/2 (TANK) are two enzymes
known to play multiple roles in cell biology, including regulation of proliferation,
differentiation and metabolism. Both of them act on the canonical Wnt/β-Catenin
pathway, but are also involved in many other independent intracellular mechanisms.
More importantly, GSK-3β and TANK have been shown to play crucial roles in different
diseases, including cancer and neurological disorders. The GSK-3β-inhibitor ‘CHIR’ and
the TANK-inhibitor ‘XAV’ are two pyrimidine molecules, holding high potential as
possible therapeutic drugs. However, their effect on neural tissue is poorly understood. In
this study, we tested the effects of CHIR and XAV on human neural precursor cells
(hNPCs) derived from human embryonic stem cells. We found that CHIR-mediated
inhibition of GSK-3β promotes neural differentiation. In contrast, XAV-mediated
inhibition of TANK leads to de-differentiation. These results highlight the relative
importance of these two enzymes in determining the neurodevelopmental status of
hNPCs. Furthermore, they shed light on the roles of Wnt signaling during early human
neurogenesis.
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INTRODUCTION
The canonical Wnt/ β-Catenin signaling pathway has been shown to play pivotal
roles in embryonic neural development as well as in adult neurogenesis [1, 2]. For
example, aberrant regulation of Wnt signaling has been proposed to underlie some of the
symptoms observed in Alzheimer’s disease, Autism and Fragile X Syndrome [3-8].
Activation of the pathway takes place when the Wnt ligand binds to its receptor,
triggering the release of β-Catenin from its inhibitory complex, which enables β-Catenin
to translocate to the nucleus, activating or repressing the expression of several target
genes [9-11]. Glycogen synthase kinase-3β (GSK-3β) and tankyrase-1/2 (TANK) are two
enzymes known to play multiple roles in cell biology, including proliferation,
differentiation and metabolism [12-15]. Although each of these enzymes is involved in
many independent intracellular mechanisms, they are both connected to the canonical
Wnt signaling pathway through regulation of β-Catenin activity, in opposite directions:
while GSK-3β activity reduces β-Catenin levels and down-regulates Wnt signaling,
TANK activity increases β-Catenin levels and up-regulates Wnt signaling. For these
reasons, much effort has been invested in the last years in developing suitable small
molecules that could inhibit or modulate the enzymatic activity of GSK-3β and TANK
[16-18]. The amino-pyrimidine CHIR99021 (‘CHIR’) is a highly specific inhibitor of
GSK-3β, with promising applications as a therapeutic drug [19]. The pyrimidine
XAV939 (‘XAV’) is a specific inhibitor of TANK and has also been successfully tested
as a promising therapeutic drug [20-22].
Currently, most studies demonstrating the importance of Wnt in neural
development have focused mainly on animal-based models and in adult neurogenesis.
Reports on the effects of CHIR and XAV so far have also been predominantly performed
on animal models, and in non-neural tissues. Recently, we reported on the molecular
mechanisms regulating impaired in-vitro neural differentiation of Fragile X Syndrome
(FXS)-human embryonic stem cells (hESCs), carrying the full mutation at the FMR1
gene [8]. In that study, we hypothesized that dysregulation of Wnt signaling could be
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responsible for the poor neural differentiation associated with these cells [23-25]. To test
that hypothesis, we applied CHIR or XAV to these cells while carrying out neuronal
differentiation. Contrary to what was reported in other FXS models, mainly adult
neurogenesis in fmr1-/- mice hippocampi [4], we observed that there was no significant
difference between FX-hESCs and controls. However, and most importantly, we did
notice that in all lines tested, CHIR increased neural differentiation while XAV decreased
it.
This interesting observation prompted us to further explore the effects of
pharmacological manipulation of Wnt on the neural fate of hESCs. Neural differentiation
of hESCs is a powerful tool in the study of neurodevelopment in a human-based in-vitro
platform [24, 26, 27]. Here, we analyzed the effects of CHIR and XAV on human neural
precursor cells (hNPCs) derived from control non-mutated hESC lines. These cells are
initially differentiated from hESCs but, under specific culture conditions, can be kept as
hNPCs for up to 10 passages [8]. During that time, they can be induced to differentiate
into neurons on-cue, by changing their culture conditions, removing basic fibroblast
growth factor (bFGF) and adding neuronal growth factors [8, 23, 24]. In this study we
induced neuronal differentiation of hNPCs, while adding CHIR or XAV. Our results
show that CHIR is a potent neuralizing agent, whereas XAV induces de-differentiation of
hNPCs. Importantly, our data are obtained from in-vitro bioassays developed specifically
for the measurement of the relative effect of these two inhibitors.
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MATERIALS AND METHODS
Human embryonic stem cell lines.
Four non-affected human embryonic stem cells (hESC) lines were used: HUES-6, HUES-
16, HUES-13 and HUES-64 [8, 28-30]. Cells were cultured on Matrigel (BD), in hESC
medium supplemented with bFGF (8ng/ml, R&D) [24]. For full information on these cell
lines, see Table 1.
In-vitro Neural Differentiation of hESCs and derivation of hNPCs.
In-vitro neural differentiation (IVND) of hESCs was carried out as previously described
[8, 24]. In brief, hESCs were grown in Neural Induction Medium (NIM) consisting of
DMEM:F12 (LifeTech.), 0.5% B27 (LifeTech.), 1% N2 (LifeTech.), 1% Glutamax
(LifeTech.), 1% Non-essential amino acids (BioInd.) and 0.1mg/ml Primocin
(InvivoGen). IVND included 3 steps: (a) formation of neuro-ectoderm aggregates in the
presence of noggin (250ng/ml, PeproTech) and bFGF (20ng/ml, R&E); (b) development
of attached neural rosettes in the presence of Shh (200ng/ml, PeproTech) and (c)
formation of neurospheres NIM supplemented with bFGF (20ng/ml, R&E). Human
Neural Precursor Cells (hNPCs) were isolated during IVND of hESCs, following
mechanical removal of neural rosettes. hNPCs were cultured on Matrigel (BD)-coated
polystyrene wells in NIM supplemented with bFGF (20ng/ml). To induce neuronal
differentiation, hNPCs were dissociated using TryplE (LifeTech.) and re-plated on Poly-
D-Lysine/Laminin (Sigma)-coated glass coverslips. NIM was changed to Neuronal
Differentiation Medium (NDM) supplemented with BDNF, GDNF and NT-3 (10ng/ml,
PeproTech). NDM consisted of Neurobasal (LifeTech.), 1% B27 (LifeTech.), 1% N2
(Life Tech.), 1% Glutamax (LifeTech.), 1% Non-essential amino acids (BioInd.) and
0.1mg/ml Primocin (InvivoGen).
Gene transcription analysis.
Relative transcription levels were analyzed by quantitative RT-PCR, as previously
described [8, 24]. RNA was extracted (RNeasy, Qiagen), reversed transcribed using
Super Script-III kit (Invitrogen), and analyzed using SYBRgreen (ABgene) in Rotor
Gene 6000 Series (Corbett). The house keeping gene GAPDH was used as a control for
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ΔΔCt analysis. All qRT-PCR assays included non-template control, non-human cells
(MEF), and human-FXS white blood cells. For all primers used see Table S1.
Immunostaining Assay.
Immunostaining was performed as previously described [8, 24]. Cells were seeded on
glass coverslips coated with Matrigel for hNPCs or with Poly-D-Lysine/Laminin for
neuronal differentiation. Cells were fixated using Cytofix (BD). Incubation with primary
antibodies was performed over-night at 4°C, detected using Cy2/Cy3-conjugated
secondary antibodies. Nuclei were stained using DAPI (Sigma). Coverslips were
mounted using Fluoromount G (Southern Biotech). Cells were imaged using an inverted
fluorescent microscope (Olympus). All conditions were similar for all lines in all
experiments. Each experiment was performed in triplicates and images were taken from 5
different fields/coverslip and >50 cells/field were analyzed by manual counting of
positively stained cells and by measurement of mean gray value of Cy2/Cy3 staining
relative to DAPI, using ImageJ software (NIH). For all antibodies used see Table S2.
Western Blot Analysis.
Western Blot analysis was carried out as previously described [8]. Protein was extracted
using reporter lysis buffer (Promega), and 25-30 µg of protein were loaded on a 7.5%
separating gel using Mini Trans-Blot Cell (Bio-Rad). Nitrocellulose membranes were
stained with primary antibodies, and detected with HRP-conjugated secondary
antibodies. Protein bends were detected using EZ-ECL (BioInd.)
Pharmacological manipulation of Wnt signaling.
The GSK3β inhibitor CHIR99021 (Tocris) and the tankyrase inhibitor XAV939
(Selleckchem) were dissolved in DMSO at 20 mM stock solutions and stored at -80°C, in
the dark. Fresh CHIR/XAV was added with every medium change, every 48 hrs.
Working concentrations of both inhibitors was 3µM in all experiments, and 0.015%
DMSO in culture medium.
Proliferation and Survival Assay.
Cells were seeded at low densities (~30,000 cells p/well in a 12-well plate) and incubated
for 7 days with either CHIR99021 or XAV939. For assessment of proliferation, cells
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were immunostained against the proliferation marker Ki-67 (R&D), and quantified
relative to DAPI staining. For survival assay, cells were manually counted using a
standard hemocytometer
Statistical Analysis:
Statistical analysis (Student’s t-test and ANOVA) was performed using SPSS, SigmaPlot
and online GraphPad QuickCalcs (http://www.graphpad.com/quickcalcs).
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RESULTS
Immediate effects of Wnt modulators on hNPCs.
In the present study we have tested the effects of CHIR, a specific GSK-3β
inhibitor and XAV, a specific TANK inhibitor, on gene expression, morphology and
differentiation potential of hNPCs lines we have previously derived from 4 different
hESC lines (Table 1).
Table 1. Full list of hESC lines used in the present study.
hESC
Line Institution Sex Sources/Information
HUES6 Harvard University XX
http://stemcelldistribution.harvard.edu/
[8, 24]
HUES16 Harvard University XY
http://stemcelldistribution.harvard.edu/
[8, 24]
HUES13 Harvard University XY
http://stemcelldistribution.harvard.edu/
[8, 23-25, 27]
HUES64 Harvard University XY
http://stemcelldistribution.harvard.edu/
[8]
Table 1: List of all hESC lines used in the present study, its parent institution, its sex and
references for further information.
The effects of CHIR and XAV on neuronal differentiation of hNPCs were
analyzed following short-term (2-3 days), mid-term (7 days), or long-term treatment (30
days) (Figure 1A).
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Figure 1. The effect of Wnt modulators on neural gene expression in hNPCs derived
from hESCs.
Figure 1. The effect of Wnt modulators on neural gene expression in hNPCs derived
from hESCs.
(A) Schematic outline of the experimental set-up of the present study.
(B) The effect of CHIR and XAV (3 µM, 2-3 days) on β-Catenin protein levels by
Western Blot analysis. β-Actin was used as a loading control. The experiment was
performed on all four hNPC lines and representative images for HUES13-hNPC line are
shown.
(C) Effect of CHIR and XAV (3 µM, 2-3 days) on the mRNA expression of neural genes.
qRT-PCR detection of GFAP and MAP2 expression (control – gray bars, CHIR – white
bars, XAV – black bars. Values are mean ± SEM. Repeated in 4 hNPC lines, n=3/line,
*p<0.05, **p<0.01, ANOVA.
First, we assessed the effect of both molecules on the levels of β-Catenin protein
in hNPCs (Figure 1B). GSK-3β phosphorylates two different serine residues on the N-
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terminus of β-Catenin, tagging it for ubiquitination and degradation. TANK indirectly
increases β-Catenin levels by de-activating AXIN, an important member of the ‘death
complex’ that degrades β-Catenin. Our results confirm that inhibition of GSK-3β by
CHIR led to a significant increase in the protein levels of β-Catenin. Conversely,
inhibition of TANK by XAV, led to a significant decrease in β-Catenin.
Next, we analyzed whether this short-termed exposure to CHIR and XAV could
change the mRNA levels of neural genes reflecting the neurodevelopmental status of
hNPCs, including GFAP, MAP2, TUJ1 and TAU (Figure 1C, Figure S1). During
formation of mature post-mitotitc neurons and glia, GFAP is reduced in neurons and
increased in glial cells, whether MAP2, TUJ1 and TAU are all increased in neurons, as
part of the dendritic and axonal cytoskeleton [26]. However, these four genes are all
expressed at the hNPC stage, where GFAP corresponds to early hNPC phenotype and
MAP2 to advanced hNPC phenotype [8, 24]. Our results show that CHIR and XAV
affected GFAP and MAP2 transcriptional levels specifically (Figure 1C), but did not
alter TUJ1 and TAU expression (Figure S1). Our results show that short-term exposure
to CHIR reduced GFAP in hNPCs (11.4±3.0% of control, p<0.01, n=3/line, 4 hNPC
lines), but it did not significantly alter the expression of MAP2. On the other hand, XAV
had opposite effects in GFAP and MAP2 expression, significantly increasing GFAP
(142.0±15% of control, p<0.05, n=3/line, 4 hNPC lines) and significantly reducing
MAP2 (12.3±9.4% of control, p<0.01, n=3/line, 4 hNPC lines). However, since GFAP in
this context is an early neural marker, we interpret the XAV-mediated increase in GFAP
as representative of reduced neurodevelopment and not enhanced glial differentiation.
Taken together, these results suggest that CHIR-mediated inhibition of GSK-3β promotes
neural differentiation, whether inhibition of TANK by XAV prevents it.
Effect of Wnt modulators on morphology, proliferation and survival of hNPCs.
To test whether CHIR induced neural differentiation and XAV inhibits it, we
subjected the hNPCs to a 7 days-long treatment with CHIR or XAV, and performed a
morphological assay which measures neural differentiation (Figure 2A). This
morphological assay is based on the premise that, as neural differentiation progresses,
cells become bigger and produce more numerous and longer projections, showing an
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increase in cellular area and perimeter. Indeed, we observed that exposing cells to CHIR
for 7 days conferred them a more neuronal phenotype, increasing the number of
projections from each cell, and especially their length (Figure 2B). In contrast, XAV
elicited the opposite effect, conferring cells with a more hESC-like primitive
morphology, lacking projections and demonstrating small round somata. Specifically,
CHIR increased the total cell area from 509.1±13.9 µm2 to 564.1±15.1 µm2 (p<0.05, >50
cells/experiment, n=3/line, 4 hNPC lines), while XAV reduced it to 282.2±9.4 µm2
(p<0.01, n same as above). Their effect on total cell perimeter was similar: CHIR
increased cellular perimeter from 106.2±2.0 µm to 120.6±1.7 µm (p<0.05) and XAV
reduced it to 71.8±1.5 µm (p<0.01).
Figure 2. The effect of Wnt modulators on neural fate.
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Figure 2. The effect of Wnt modulators on neural fate.
(A) Schematic presentation of the morphological changes undergoing hESCs during in-
vitro neural differentiation, and how they are reflected in the relative change in cell area
and perimeter.
(B) Morphological changes observed in hNPCs following treatment with CHIR or XAV
(3 µM, 7 days). The analysis was repeated in all 4 hNPC lines, representative images are
shown for of HUES64-hNPCs (left panel). Morphological analysis (right panel) of total
area (µm2) and total perimeter (µm) carried out for the effect of CHIR (white bars) or
XAV (black bars) as compared to control (grey bars). Values are mean ± SEM. Repeated
in 4 hNPC lines, n=3/line, *p<0.05, **p<0.01, ANOVA.
(C-D) Proliferation and survival of hNPCs following exposure to CHIR and XAV (3
µM, 7 days). Cells were stained for DAPI (nuclear staining, blue) and the proliferation
marker Ki67 (red). The experiment was repeated in all 4 hNPC lines, representative
images shown for HUES6-derived hNPCs. Proliferation was measured as the number of
Ki67-positive cells relative to DAPI staining. Survival rate was established by manual
count of cells using a standard hemocytometer. Proliferation and survival rate are shown
as % of control (control – gray, CHIR – white, XAV – black). Values are mean ± SEM.
Repeated in 4 hNPC lines, n=3/line, *p<0.05, **p<0.01, ANOVA.
GSK-3β and TANK have a profound effect on regulation of cell proliferation, by
regulation of the Wnt/β-Catenin pathway, but also in a Wnt-independent manner.
Furthermore, it has been well established that differentiation reduces proliferation, and
indeed terminally-differentiated cells, such as adult neurons, are post-mitotic.
Correspondingly, undifferentiated or de-differentiated cells, like hESCs or cancer cells,
have high rates of proliferation. Therefore, we examined how a 7 days-long exposure to
CHIR or XAV could affect proliferation and survival of hNPCs. Quantitative analysis of
the expression of the proliferation marker Ki67 showed that treatment with CHIR
resulted in a significantly reduced proliferation rate, whereas treatment with XAV did not
(Figure 2C). Indeed, the expression of Ki67 was 37.3±3.1% of that found in control
hNPC cultures after 7 days of continuous treatment with CHIR (p<0.01, >50
cells/experiment, n=3/line, 4 hNPC lines), but it did not significantly differ from the
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levels of Ki67 expression in hNPC cultures subjected to treatment with XAV. As a means
to further confirm this, we performed a survival assay in which cells were seeded at very
low densities and treated with CHIR or XAV at the same conditions mentioned before
(Figure 2D). This assay confirmed that CHIR evokes a robust reduction in cell survival
(p<0.01). This assay also showed that XAV had a statistically significant effect as well
(p<0.05), however it was less pronounced than the effect of CHIR. Taken together, these
data suggest that CHIR acts as a neuralizing agent in hNPCs, increasing neural
differentiation and reducing proliferation. In contrast, XAV acts as a de-differentiation
agent, without significantly altering the cell proliferation rate.
Effect of Wnt modulators on prolonged neuronal differentiation of hNPCs.
In order to assess how CHIR and XAV may affect neurogenesis, we induced
neuronal maturation in hNPCs and tracked their development for a total of 30 days. The
relative increase or decrease in MAP2 and GFAP, is a quantitative tool to directly
measure the efficacy of neurogenesis in-vitro, as we and others have previously shown
[8, 24]. Therefore, we analyzed the relative protein levels of MAP2 and GFAP using
immunostaining assays (Figure 3A,B).
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Figure 3. Effect of Wnt modulators on neuronal differentiation of hNPCs.
Figure 3. Effect of Wnt modulators on neuronal differentiation of hNPCs.
(A) Representative images of hNPCs immunostained for MAP2 (red) before beginning of
neuronal differentiation (“Day 0”) and following 30 days of neuronal differentiation
(“Day 30”). Repeated in all 4 hNPC lines, representative images shown for HUES16
hNPC line.
(B-C) Effect of CHIR and XAV (3 µM, 30 days) on neuronal differentiation of hNPCs.
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(B) Quantification of MAP2 and GFAP expression relative to DAPI at day 30 of neuronal
differentiation of hNPCs (control – gray, CHIR – white, XAV – black).
Values are mean ± SEM. Repeated in 4 hNPC lines, n=3/line, *p<0.05, ANOVA.
(C) Relative change in area of cell soma and total cell perimeter at day 30 of neuronal
differentiation of hNPCs (control – gray, CHIR – white, XAV – black). Values are mean
± SEM. Repeated in 4 hNPC lines, n=3/line, *p<0.05, **p<0.01, ANOVA.
Our results show that by day 30 CHIR did not significantly increase the levels of
MAP or GFAP. However, XAV significantly reduced the expression of both markers
(MAP2 64.5±5.6% of control, GFAP 75.2±4.6, p<0.05, >50 cells/experiment, n=3/line, 4
hNPC lines). Since the effect of CHIR was not reflected in these assays, we analyzed
morphological alterations induced by both molecules, as previously described (Figure
2A,B). Since the main morphological hallmark of neuronal differentiation is the
development of long and thin neurites, we measured the total area only for somata,
whereas the perimeter was measured for the whole cell, including neurites (Figure 3C).
Our results show that, after 30 days of treatment, CHIR did not significantly affect the
size of cell bodies (135.6±13.7% of control, p>0.05, >50 cells/experiment, n=3/line, 4
hNPC lines), but significantly increased their perimeter, reflecting the increase in
projection number and length (131.5±9.8% of control, p<0.05). Conversely, XAV
robustly reduced both the area of the somas and the total perimeter of cells (area:
31.7±11.4% of control; perimeter: 38.8±6.2% of control, p<0.01). These results confirm
the neuralizing effect of CHIR and the de-differentiation effect of XAV during the time-
course of neuronal differentiation. All of these measurements were carried-out also at
days 10 and 20 of neuronal differentiation, with no significant effects found in neither of
those two time-points (data not shown). This suggests that during the initiation of
neuronal maturation the activity of GSK-3β and TANK is probably redundant with other
molecular mechanisms, and only at later stages they become pivotal in the process.
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DISCUSSION
From the results we have shown in this study we conclude that GSK-3β inhibition
by CHIR promotes neural differentiation in hNPCs. Several studies have shown that
CHIR induces differentiation into the neural lineage in hESCs, as well as in human
induced pluripotent stem cells (hiPSCs) [31-35]. The same was true in primate-derived
ESCs and iPSCs [36]. However, in rodent-based models, this was inconsistent. Indeed,
two studies reported that in rat-derived ESCs and in mouse embryonic fibroblast, CHIR
promoted neural differentiation similar to the human model [37, 38], but two other
studies showed that in mouse ESCs CHIR inhibits differentiation, enhancing
pluripotency-maintenance mechanisms [39, 40]. This discrepancy between human and
murine in-vitro platforms, highlights the importance of the use of human-based models in
neurodevelopmental research. Also, it emphasizes the pleiotropic activity of GSK-3β,
which can play many different functions that are species and tissue-specific, and depend
on developmental status.
In this report we have also shown that XAV induces de-differentiation of hNPCs
towards a more primitive developmental stage. However, to the best of our knowledge,
no other similar reports have been produced so far, in which the effect of XAV was
analyzed in hNPCs derived from hESCs or hiPSCs. In a human neurobolastoma cell line
XAV was found to induce apoptosis by blocking Wnt signaling [41]. In zebrafish
embryos, XAV inhibited retinal development [42]. Using mouse Epiblast-derived stem
cells, XAV increased the development of forebrain precursors in one report [43], but
inhibited differentiation and enhanced pluripotency in another report [44]. Finally, in
mESCs, it was shown that XAV promotes differentiation into cardiomyocytes [45].
These discrepancies could be attributed to the model used in each study, and to whether
in those tissues TANK activity affects the regulation of the Wnt signal only, or not is
involved in other currently unknown mechanisms. Here too, similar to GSK-3β, the
pleiotropy of TANK activity could lead to mixed interpretations of the results
Our data suggest that the neuralizing effect of CHIR and the anti-neuralizing
effect of XAV on hNPCs, is probably due to their involvement in the Wnt signaling
pathway and their opposite effect on β-Catenin levels. This is because CHIR and XAV
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had opposite effects on cell proliferation as well as on neural differentiation. However,
more research is needed to determine which specific functions of GSK-3β and TANK are
Wnt-dependent and which are Wnt-independent in the regulation of hNPCs
developmental fate.
Finally, we have shown here that hNPCs derived from hESCs are a valuable tool
for in-vitro human-based drug screening studies. As briefly reviewed here, there are
striking discrepancies between human-based in-vitro models and animal-based models.
Finding the molecular basis for these differences is crucial to improve basic knowledge
as well as to improve future therapeutic strategies.
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was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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The copyright holder for this preprint (which. http://dx.doi.org/10.1101/509638doi: bioRxiv preprint first posted online Jan. 1, 2019;
.CC-BY-NC 4.0 International licenseIt is made available under a
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which. http://dx.doi.org/10.1101/509638doi: bioRxiv preprint first posted online Jan. 1, 2019;
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was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which. http://dx.doi.org/10.1101/509638doi: bioRxiv preprint first posted online Jan. 1, 2019;
.CC-BY-NC 4.0 International licenseIt is made available under a
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which. http://dx.doi.org/10.1101/509638doi: bioRxiv preprint first posted online Jan. 1, 2019;
.CC-BY-NC 4.0 International licenseIt is made available under a
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which. http://dx.doi.org/10.1101/509638doi: bioRxiv preprint first posted online Jan. 1, 2019;
