Competence to epithelialise coincides with competence to differentiate in pluripotent cells

TL;DR: Qualitative imaging approaches are used to measure cellular rearrangements that accompany exit from naive pluripotency and conclude that competence to epithelialise is actively regulated and linked to differentiation-competence through the transcription factor Tcf15.

Abstract: Summary Pluripotent cells reorganise themselves into an epithelium before they initiate differentiation, but it is not clear how these two events are mechanistically linked. Here we use quantitative imaging approaches to measure cellular rearrangements that accompany exit from naive pluripotency. We show that competence to epithelialise, like competence to differentiate, is a regulated process. The pro-differentiation transcription factor Tcf15 prospectively identifies cells that are competent to epithelialise. We identify early upregulation of the laminin receptor integrin alpha3 prior to differentiation and show that Tcf15 helps to regulate this change. Finally, we show that Tcf15 identifies and is required for efficient differentiation of a primed subpopulation of pluripotent cells. We conclude that competence to epithelialise is actively regulated and linked to differentiation-competence through the transcription factor Tcf15.

Summary

Introduction

• A constitutively activated (BMP-resistant) form of Tcf15 has pro-differentiation activity in pluripotent cells (Davies et al, 2013), but the mechanism by which Tcf15 promotes differentiation has not been reported.
• The authors show that although naive pluripotent cells can polarise they are not fully competent to epithelialise in response to matrix, whereas primed pluripotent cells can efficiently form an epithelium.
• These two parallel processes are coordinated in part by the transcription factor Tcf15, analogous with the role of Tcf15 to coordinate epithelialisation and differentiation in the somites (Burgess et al, 1996).

Results

• Pluripotent cells co-align into an epithelium as they become prepare to differentiate.
• The authors used this approach to assess the extent to which pluripotent cells are organised into an epithelium before and after implantation of the embryo.
• The authors next confirmed that changes in expression of Itga3 follow a similar pattern to changes in expression of Tcf15 over the course of EpiSC differentiation (Fig 3D).
• Error bars represent the standard deviation of 2 independent clones with 3 technical replicates each.
• These cells have considerably reduced levels of Tcf15 protein (Supp Fig 2D), but were still able to detect a differentiation-advantage in the Tcf15high subpopulation compared to the Tcf15-low subpopulation, as previously observed in their geneconserving Tcf15 reporter cells (Fig 5D).

Discussion

• Amniotes organise their pluripotent cells into an epithelium as they prepare to gastrulate, suggesting that this morphological event may be an important prerequisite for the formation of the body plan (Sheng, 2015).
• The extrinsic events that drive epithelialisation are well described (Li et al, 2003), however the intrinsic changes that enable this process are less well understood.
• This raises the question of whether Tcf15's influence on adhesion and differentiation is regulated independently or whether adhesive changes might reinforce pro-differentiation signals.
• There has been a great focus on the transcriptional and epigenetic events that prepare pluripotent cells to differentiate, but less appreciation of changes in adhesion and morphology that could influence differentiation (Malaguti et al, 2013; Livigni et al, 2013).
• While these changes in adhesion and morphology are unlikely to be dominant drivers of differentiation, they are likely to influence the efficiency with which cells can respond to differentiation cues and may be important for making development more robust.

Cell lines

• Cell lines made in this study were generated from mouse ES cell line E14tg2a (Smith & Hooper, 1987).
• 1 µg/mL of doxycycline was used to induce expression of the transgene.
• Cell line 216D1 is a Tcf15 reporter cell line which a gtx-IRES-Venus cassette was incorporated into the Tcf15 locus downstream of the coding sequence (Tanaka et al, 2008).
• This cell line preserves both coding alleles.

Cell culture

• LIF+FCS culture of ES cells: Glasgow Modified Eagle's Medium (GMEM), 10% foetal calf serum, 1 mM sodium pyruvate, 1x non-essential amino acids, 2 mM L-glutamine, 0.1 mM 2- mercapthoethanol and 100 units/mL LIF.
• EpiLC differentiation was performed as previously described (Hayashi et al, 2011) and EpiSC differentiation was performed as previously described (Guo & Smith, 2010).
• Neural differentiation was performed in N2B27 media on gelatin as previously described (Pollard et al, 2006).
• Laminin was used at 5 µg/mL in PBS and dishes were coated for overnight at 4oC.

Targeting construct for Tcf15

• The targeting construct for Tcf15 contained a 2.3kb 5' homology arm and a 2.4kb 3' homology arm corresponding to the sequences flanking the first coding exon of Tcf15.
• Within the two homology arms is the coding sequence for Venus followed by an frt-flanked pgk-neo MC1-TK cassette selection cassette.
• This cassette was removed by transfection of pFlpO after isolation of successfully targeted clonal cell lines.
• A PGK-DTA cassette was placed upstream of the 5' homology arm to enable negative selection of clones in which the targeting cassette had randomly integrated.

Southern blotting

• Genomic DNA was extracted from mouse ES cells using the DNeasy Blood and Tissue kit .
• Southern blotting was performed as previously described (Southern, 1975).

Transcriptome analysis

• For RNAseq analysis total RNA was isolated from cells using the Absolutely RNA Miniprep Kit and RNA quality was verified using an 2100 Bioanalyzer.
• Subsequent cDNA library preparation, sequencing and bioinformatics analysis, including differential gene expression analyses, were performed by the Edinburgh Genomics facility.
• Gene ontology analysis was performed on genes upregulated at 24h using the STRING database (Szklarczyk et al, 2017).
• CRNA quality was checked using an Agilent 2100 Bioanalyser and hybridization was performed on a MouseWG6 v2 BeadChip .
• After 7 days, alkaline phosphatase staining was performed using the Leukocyte Alkaline Phosphatase Kit .

Fluorescence Activated Cell Sorting

• Cells were dissociated into single cell suspensions in ice-cold PBS+10% FCS, in the presence of either 100ng/ml DAPI or 1µg/ml propidium iodide to stain dead cells.
• Cell sorting was performed on a BD FACSAria.
• Cells were also gated for immunoreactivity to PECAM in order to exclude differentiated (PECAM-negative) cells.
• Embryo collection and chimera generation Pre- and peri-implantation embryos were obtained by flushing uteri with a large-bore blunted needle in M2 medium .
• For generating chimeric mice, F1 female mice were superovulated (100 IU/ml PMSG, ProSpec, and 100 IU/ml HCG, Intervet, intraperitoneal injections 48h apart) and crossed with wild-type stud male mice.

Western blotting

• Cells were lysed in RIPA buffer + 1X PMSF (Alpha Diagnostics).
• 20µg protein lysates were run on 4-12% NuPage Bis-Tris Gel and transferred onto Amersham Hybond ECL Nitrocellulose Membrane (GE Healthcare).
• Membranes were blocked in 5% Amersham ECL Prime Blocking Agent (GE Healthcare) + 0.1% Tween 20 in PBS.
• Membranes were incubated in primary antibody overnight at 4°C, washed 3 times in PBS + 0.1% Tween 20, incubated in HRP-conjugated secondary antibody for 1 hour at room temperature and washed 3 times in PBS + 0.1% Tween 20.
• The Tcf15 antibody used for Western blotting was obtained from Santa Cruz (sc46438) and used at a dilution of 1:100.

Immunofluorescence

• Samples were fixed in 4% formaldehyde for 10 min at room temperature (cells and blastocysts) or for 30 min at 4°C (E6.5 embryos).
• Antibodies were all diluted in blocking solution.
• Nuclei were counter-stained with Dapi except when samples were used for image analysis.

Imaging

• Pluripotent cells were imaged with a Leica SpE inverted confocal microscope using an ACS APO 63X objective with oil immersion and NA=1.3.
• Images were further analysed using PickCells (https://pickcellslab.frama.io/docs/).
• The polarity angle was defined as the angle between the cell’s polarity vector and the vector normal to the embryo cavity.

Figures

Figure 5: Tcf15 identifies cells that are primed to differentiateA-B: 216D1 Tcf15-Venus reporter ES cells were sorted into Venus-high (top 30%) or Venus-low (bottom 30%) and plated into N2B27 to initiate neural differentiation. Samples were taken and analysed by qPCR each day for the next four days then analysed by qPCR for the stated markers (A) or stained for TUJ1 on day 4 (B). C 216D1 Tcf15-Venus reporter ES cells were sorted into Venus-high (top 30%) or Venus-low (bottom 30%) and plated at low density into LIF+FCS. Colonies were allowed to form over 6 days then the culture was fixed and analysed for alkaline phosphatase activity. D: Heterozygous or null Tcf15-VenusKI cells were sorted into Venus-high (top 30%) or Venus-low (bottom 30%) and plated into N2B27 to initiate neural differentiation. Samples were taken and analysed by qPCR each day for the next five days. Two different Tcf15 null clones were analysed: A1 and A2.

Figure 3: Itga3 is upregulated in Tcf15+ cells during the transition from naive to primed states

Figure 1: Pluripotent cells co-align into an epithelium as they prepare to differentiate

Figure 4: Tcf15 regulates changes in adhesion at the onset of differentiation

Figure 2: Tcf15 identifies cells that are competent to epithelialise

Full text

Competence to epithelialise coincides with competence to differentiate
in pluripotent cells
Chia-Yi Lin
1
†
, Tulin Tatar
1†
, Guillaume Blin
1
, Mattias Malaguti
1
, Rosa Portero Migueles
1
, Hongyu
Shao
1
, Naiming Chen
1
, Ian Chambers
1
, Sally Lowell
1
*
1
MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences,
University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU.
†
Equal contribution
*Author for correspondence sally.lowell@ed.ac.uk
Summary
Pluripotent cells reorganise themselves into an epithelium before they initiate differentiation, but it
is not clear how these two events are mechanistically linked. Here we use quantitative imaging
approaches to measure cellular rearrangements that accompany exit from naive pluripotency. We
show that competence to epithelialise, like competence to differentiate, is a regulated process. The
pro-differentiation transcription factor Tcf15 prospectively identifies cells that are competent to
epithelialise. We identify early upregulation of the laminin receptor integrin alpha3 prior to
differentiation and show that Tcf15 helps to regulate this change. Finally, we show that Tcf15
identifies and is required for efficient differentiation of a primed subpopulation of pluripotent cells.
We conclude that competence to epithelialise is actively regulated and linked to differentiation-
competence through the transcription factor Tcf15.
Introduction
There is an increasing appreciation that morphological changes are not simply a consequence of
differentiation but rather the two processes are often reciprocally interlinked (Gilmour et al, 2017;
Chan et al, 2017). For example, in all amniotes pluripotent cells organise themselves into an
epithelium prior to gastrulation (Nowotschin & Hadjantonakis, 2010; Bedzhov & Zernicka-Goetz,
2014; Sheng, 2015), and it has been suggested that this morphological change may be a
prerequisite for subsequent lineage commitment (Ranga et al, 2014). At the same time, major
changes in gene expression set up a new transcriptional state that makes pluripotent cells
competent to differentiate (Nichols & Smith, 2009; Buecker et al, 2014; Boroviak et al, 2015). It is
not understood whether the process of epithelialisation is mechanistically linked to differentiation-
competence.
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Pluripotent cells polarise before they commit to a primed state (Shahbazi et al, 2017). Polarisation
is initiated by integrin-mediated adhesion to the laminin matrix secreted by the emerging
extraembryonic endoderm (Bedzhov & Zernicka-Goetz, 2014; Li et al, 2016) and subsequently
stabilised by PTEN (Meng et al, 2017) and Integrin-linked-kinase (Sakai, 2003). However, several
open questions remain. Is the initiation of epithelialisation a spontaneous response to matrix or a
regulated process? If epithelialisation is a regulated process, is it coordinated with differentiation or
are the two processes regulated independently? What are the transcription factors and adhesion
molecules that influence competence to epithelialise and help coordinate it with differentiation?
Tcf15 is a bHLH transcription factor that regulates epithelialisation of the somites (Burgess et al,
1996). It is also expressed in a subset of the Nanog-low subpopulation of mouse embryonic stem
cells and within the inner cell mass of pre-implantation embryos (Davies et al, 2013). Tcf15 is
activated transcriptionally by FGF and Wnt signalling (Linker et al, 2005; Davies et al, 2013). Its
activity is suppressed post-translationally by BMP signalling: BMP upregulates Id1, which
sequesters the E-proteins that form essential heterodimerisation partners with Tcf15 (Wilson-
Rawls et al, 2004). A constitutively activated (BMP-resistant) form of Tcf15 has pro-differentiation
activity in pluripotent cells (Davies et al, 2013), but the mechanism by which Tcf15 promotes
differentiation has not been reported. Tcf15 null embryos survive through gastrulation (Burgess et
al, 1996) but it is not known whether Tcf15 contributes to morphogenesis or differentiation during
exit from naive pluripotency.
Here, we use quantitative image analysis to measure emergence of epithelial organisation during
exit from naive pluripotency in vivo and in vitro. We show that although naive pluripotent cells can
polarise they are not fully competent to epithelialise in response to matrix, whereas primed
pluripotent cells can efficiently form an epithelium. Given that Tcf15 is expressed as cells
downregulate Nanog (Davies et al, 2013) and that Tcf15 is required for epithelialisation of the
somites (Burgess et al, 1996) we speculated that it may regulate epithelialisation of pluripotent
cells. We use Tcf15 reporters and gain/loss of function approaches to show that Tcf15 identifies
cells that are primed to epithelialise and that Tcf15 regulates the timely emergence the alpha
subunit of the laminin receptor integrin α 3β1. We also find that Tcf15 is required for efficient
differentiation of a primed subset of pluripotent cells.
We conclude that, competence to epithelialise is an actively regulated process that is initiated in
parallel with, rather than as a consequence of, exit from naive pluripotency. These two parallel
processes are coordinated in part by the transcription factor Tcf15, analogous with the role of
Tcf15 to coordinate epithelialisation and differentiation in the somites (Burgess et al, 1996).
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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Results
Pluripotent cells co-align into an epithelium as they become prepare to differentiate
We developed quantitative imaging tools for measuring the emergence of epithelial organisation
based on segmenting individual nuclei and centrosomes and measuring nucleus centrosome
vectors relative to the cavity of the embryo (Fig 1A) (Burute et al, 2017)
https://pickcellslab.frama.io/docs/use/features/segmentation/nc_assignments/. We used this
approach to assess the extent to which pluripotent cells are organised into an epithelium before
and after implantation of the embryo. We confirm that pluripotent cells in the blastocyst have no
apparent epithelial organisation. Nucleus-centrosome vectors measured relative to the cavity show
no trend towards any particular direction: the number of cells pointing towards the cavity (angle
<90
o
C) is similar to the number of cells pointing away from the cavity (angle >90
o
C) (Fig 1 B-C). In
contrast, in the postimplantation epiblast almost all pluripotent cells point towards the cavity
(angles <90
o
C) (Fig 1 B,D). These measurements confirm that pluripotent cells tend to co-align into
an epithelial organisation during peri-implantation development, as expected (Nowotschin &
Hadjantonakis, 2010).
Competence to epithelialise in response to matrix is acquired as pluripotent cells prepare to
differentiate
In the embryo, laminin matrix is laid down by the emerging extraembryonic endoderm from late
postimplantation development (Li et al, 2003). This raises the question of whether the formation of
the pluripotent epithelium is a spontaneous response to the appearance of this extracellular matrix
or whether it requires actively regulated intrinsic changes.
Formation of an epithelium involves multiple steps including acquisition of polarity, an increase in
cell-matrix adhesion, and formation of a lumen, each of which could be independently regulated. It
has previously been reported that naive pluripotent cells are able to polarise but unable to form a
lumen (Shahbazi et al, 2017) and that polarisation occurs prior to neural differentiation (Ranga et al,
2014). In keeping with these reports, we confirm that naive cells do not efficiently form stable 3D
cysts when cultured in matrigel but become competent to do so after 48h of neural differentiation in
N2B27 (Supp Fig S1).
In order to analyse epithelialisation independently of luminogenesis, we turned to a 2D culture
system. We analysed nucleus-centrosome vectors relative to the bottom of the culture dish (Fig
1E). We first analysed pluripotent cells grown in 2iLIF (Fig1F), which are equivalent to pluripotent
cells of the preimplantation embryo (Boroviak et al, 2015) and compared these with epiblast stem
cells (Fig 1G), which are equivalent to pluripotent cells of the postimplantation epiblast (Tesar et al,
2007; Brons et al, 2007). Each of these two cell types adopted an organisation similar to their
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Figure 1: Pluripotent cells co-align into an epithelium as they prepare to differentiate
A-D: Cell polarity analysis in vivo. A: Schematic indicating the measurement of an angle between the
nucleus to centrosome vector of a cell relative to the cavity of the embryo.
B: Beeswarm-boxplot showing the distribution of angles between the nucleus and centrosome vector of the
cell and its vector normal to the embryo cavity. Angles are shown for ICM cells in E3.5 blastocysts or for
cells of the epiblast in E6.5 embryos. Angles are shown for ICM cells in E3.5 blastocysts (10 blastocysts for
a total of 130 ICM cells) or for cells of the epiblast in one E6.5 embryo (354 cells).
C-D: Representative 3D rendering of the processed images are given on each side of the plot. A shows an
E3.5 blastocyst with and B shows an E6.5 embryo (transverse view). The nuclei of pluripotent cells are
colored in cyan, extra-embryonic cell nuclei in grey, centrosomes in magenta and nucleus-centrosome
vectors in yellow.
E:H: Cell polarity analysis in pluripotent stem cell cultures. E: Schematic indicating the measurement of an
angle between the nucleus to centrosome vector of a cell relative to the bottom of the dish
F-G Rose diagrams show the distribution of angles between the nucleus to centrosome vector and its vector
normal to the bottom of the dish. The green line in rose diagrams indicate the average angle, and the red arc
represents the standard deviation of the angle. N indicates the number of cells included in the analysis. A
representative confocal image is given for each culture condition stained for Zo1 to indicate AP polarity and
E-Cadherin to demarcate cell outlines. ICM: inner cell mass
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in-vivo equivalents: 2iLIF cells fail to co-align while EpiSC co-align into an epithelial organisation
with nucleus-centrosome vectors pointing up away from the surface of the dish (Fig 1F, G). These
measurements confirm that cells co-align during the transition from naive to primed states in
culture, as they do in vivo.
We then analysed cells cultured in LIF+FCS, where cells can exist in a mixture of naive and primed
states within the same environmental conditions. Analysis of nucleus-centrosome vectors
confirmed that, unlike naive cells, a majority of cells are aligned into an epithelium with centrosome
pointing away from the bottom of the dish, but unlike primed cells a significant minority do not
adopt this epithelial organisation (Fig1H): LIF+FCS). We conclude that pluripotent cells do not
become fully competent to epithelialise until they reach a primed state.
Figure 2: Tcf15 identifies cells that are competent to epithelialise
A: A typical colony of 216D1 Tcf15-Venus reporter ES cells expanded from a single cell in LIF+FCS for 6
days showing heterogeneous expression of Tcf15-Venus
B: 216D1 Tcf15-Venus ES cell were cultured in LIF+FCS then sorted by FACS for Venus-high (top 30%) and
Venus-low (bottom 30%) before plating on laminin for 24h in N2B27 media. Venus-high cells form flat
epithelial colonies while Venus-low cells form domed colonies.
C: 216D1 Tcf15-Venus ES cell were cultured in LIF+FCS then sorted by FACS for Venus-high (top 30%) and
Venus-low (bottom 30%) before being subjected to microarray analysis: the top 20 differentially expressed
genes between the two populations are listed here. Genes associated with adhesion and morphology are
highlighted in red text.
D: qPCR analysis of gene expression in FACS sorted Tcf15-Venus-high and Tcf15-Venus-low populations.
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Frequently asked questions

Q1. What are the contributions in "Competence to epithelialise coincides with competence to differentiate in pluripotent cells" ?

The authors show that competence to epithelialise, like competence to differentiate, is a regulated process. The authors identify early upregulation of the laminin receptor integrin alpha3 prior to differentiation and show that Tcf15 helps to regulate this change. Finally, the authors show that Tcf15 identifies and is required for efficient differentiation of a primed subpopulation of pluripotent cells.