Experiments described above unveiled that Jmjd2c is not required for the generation and maintenance of extra-embryonic endoderm and epiblast stem cells. Moreover, Jmjd2c-depleted cells exhibit a higher propensity to differentiate towards extra-embryonic endoderm lineages, as notably suggested by the rapid induction of PrE markers upon atRA treatment of Jmjd2c-KO ESCs (Fig.4.1), and by skewed differentiation into extra-embryonic endoderm-like cells upon cEpiSC mesoderm induction (Fig.4.5). To further test whether Jmjd2c might impact on the balance between PrE and epiblast lineage segregation, WT and Jmjd2c-KO ESCs were induced to differentiate upon hanging-drop embryoid body (HD-EB) formation. Unlike classic embryoid body assays, static suspension of EBs formed by low number of cells here facilitates the synchronised emergence of PrE- and epiblast-like populations in a 'salt-and-pepper' manner, which are subsequently segregated into an organized outer (PrE-like) layer and an inner (epiblast-like) population (Fig.4.6A), hence mimicking the early PrE/epiblast segregation events seen in the developing blastocyst (Lavial et al., 2012; Rula et al., 2007). A comprehensive optimization was initially preformed for drop volume, cell number, and timing of differentiation. About 500 ESCs were used per drop to induce the formation small aggregates in the presence of reduced serum level and in the absence of LIF, over 5 days. Expression of epiblast (Oct4, Fgf5) and PrE (Gata4, Gata6) markers was quantified over the course of the experiment by RT-qPCR (Fig.4.6B). Additionally, day 5 HD-EBs were fixed and stained for Oct4 and Gata4, in order to delineate the formation of the two inner (epiblast-like) and outer (PrE-like) EB layers, respectively (Fig.4.6C). Despite careful optimization this approach proved to be technically challenging and only two independent experiments out of 10 performed provided a sufficient amount of material for transcript quantification (Fig.4.6B). Here, overall down-regulation of Oct4 was observed in WT HD-EBs, in contrast to Jmjd2c-KO HD-EBs that retained partial Oct4 expression at day 5 of differentiation, as previously found in late Jmjd2c-KO EBs (Section 3.2.4). Fgf5 expression was induced from day 3 onwards and the early PrE marker Gata6 highly up- regulated by day 5 in both cell populations. (Fig.4.6B). Interestingly, the expression of the late PrE
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maker Gata4 was also prominently induced but in Jmjd2c-KO HD-EBs only (Fig.4.6B). Gata4 is a known Gata6 target gene, indicative of PrE maturation rather than specification (Frankenberg et al., 2011), hence suggesting that PrE maturation might be favoured or accelerated in HD-EBs in the absence of Jmjd2c. However, in the few successful Gata4 staining experiments performed on whole HD-EBs, no striking prevalence for the formation of an outer layer could be identified in Jmjd2c-KO HD-EBs (Fig.4.6C).
Figure 4.6 Induction of hanging-drop embryoid bodies (HD-EBs) is insufficient to uncover a bias in cell fate commitment in Jmjd2c-knockout ESCs
A. Schematic representation of hanging-drop embryoid body (HD-EB) assay, mimicking the early
segregation events in the blastocyst. Formation of cell aggregates from low number of cells in suspension promotes the synchronised emergence of primitive-endoderm (PrE)- and epiblast(Epi)-like populations in a 'salt-and-pepper' manner, which are subsequently segregated into and organized outer (green) an inner (red) layer, respectively. B. Transcript levels of epiblast (Oct4, Fgf5) and PrE markers (Gata6, Gata4), during induction of HD-EBs from WT (ESCWT) and KO (ESCJmjd2c-KO) ESCs. Data were normalized to housekeeping
genes (S17, L19) and represented as average of 2 independent experiments ±SD. C. Immunofluorescence labelling of Oct4 and Gata6, for identification of Epi-like and PrE-like (arrows) layers respectively in HD-
DAPI Oct4 Gata4 Oct4 Gata4
ES C Jm jd 2c -KO ES C WT PrE-like Epi-like A. C. 0.0 0.5 1.0 0 3 5 Gata4 0.0 2.0 4.0 0 3 5 Gata6 0.0 2.0 4.0 0 3 5 Fgf5 0 20 40 0 3 5 Oct4
ESCWT ESCJmjd2c-KO
Time/days after EpiSC conversion
m R NA le ve ls B.
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EBs induced from WT and Jmjd2c-KO ESCs at day 6. Bars, 100µm. Results in B and the HD-EBs used to generate the results in C were produced by Donja Karimlou.
The unforeseen variability between experiments suggested that this approach might not be fully reliable to uncover a bias towards the extra-embryonic PrE in Jmjd2c-KO ESCs. Moreover, the quality of immunofluorescent staining could have been greatly improved by alternative approaches such as staining of sectioned agarose-embedded EBs (Lavial et al., 2012).
As a different approach to test whether Jmjd2c-KO ESCs might preferentially be allocated into the outer PrE-like layer as opposed to the inner epiblast-like layer, labelled WT and Jmjd2c- KO ESCs were mixed in a 50:50 ratio, and induced to form chimeric HD-EBs (Lavial et al., 2012). For this, WT ESCs were first labelled with a red fluorescence protein by transfection with a piggyBac vector carrying a CAG promoter driven dsRed transgene (CAG-dsRed*MST). Moreover, to ensure that the level of GFP produced by the genetrap cassette in Jmjd2c-KO ESCs would not be a limitation, and to guarantee that both ESC lines would be subjected to the same manipulations, KO ESCs were also transfected with a piggyBac vector carrying a CAG promoter driven GFP (CAG-GFP). These vectors were kindly provided by Dr Jennifer Nichols (Cambridge University). Thus, both cell populations were cultured for at least 4 passages under puromycin selection before induction of HD-EBs, and the levels of fluorescence produced by these vectors was confirmed under ESC culture conditions (Fig.4.7A). An equal ratio of WT (red) and KO (GFP) ESCs was mixed and 500 cells were used per drop in this assay (Fig.4.7A). The distribution of GFP and Red fluorescence was determined by confocal microscopy up to 7 days after HD-EB formation (Fig.4.7B). Both Green and Red signals appeared however, evenly distributed throughout the analysed aggregates, showing no striking evidence for preferential assignment of Jmjd2c-KO ESCs to the PrE layer (Fig.4.7B).
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Figure 4.7 Generation of chimeric hanging-drop embryoid bodies
A. Schematic representation of generation chimeric hanging-drop embryoid body (HD-EB) with equal
number of labelled WT (dsRed) and Jmjd2c-KO (GFP) ESCs. B. Immunofluorescence signals for WT (red) and Jmjd2c-KO (green) ESCs upon HD-EB formation at day 5, 6, and 7, showing no preferential location of labelled cells in the outer (PrE) and inner (epiblast) parts of chimeric HD-EBs. Bar, 100µm.
IV.3 Summary and Conclusions
Collectively, experiments described in Chapters III and IV uncover that Jmjd2c-KO ESCs fail to execute multi-lineage, epiblast-derived differentiation upon EB formation. Instead, these cells were found to preferentially adopt an extra-embryonic endoderm-like phenotype during the early steps of germ layer differentiation as further unveiled upon mesodermal induction of converted Jmjd2c-KO EpiSCs. This corroborates with a prominent and rapid induction of a PrE- like phenotype upon atRA treatment of Jmjd2c-depleted ESCs. Moreover, these cells also proved capable to stably convert into self-renewing XEN cell-like populations. However, no evidence could be obtained to suggest that Jmjd2c might directly impact in PrE and epiblast lineage segregation in the early embryo, as investigated here in vitro upon hanging-drop EB formation.
A. WT KO DAPI WT KO DAPI WT KO D ay 7 Da y 6 Da y 5 B.
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Moreover, the persistent expression of the Epiblast marker Fgf5 upon EB induction, and the EpiS-like phenotype adopted upon neuronal progenitor induction, uncovered in Chapter III, suggested that Jmjd2c-KO differentiation was blocked at an epiblast-like stage. To validate this assumption, the ability of Jmjd2c-KO ESCs to be converted into EpiSCs was tested. Interestingly, converted EpiSCs were established, acquiring however an immature phenotype in the absence of Jmjd2c. Testing the differentiation capacity of this “immature” cell population upon mesoderm induction uncovered a differentiation bias towards an extra-embryonic endoderm like phenotype.
Altogether, these experiments established that Jmjd2c is strictly required for proper somatic differentiation after an epiblast-like stage, but dispensable for the emergence of an extra- embryonic phenotype. The molecular basis for the impaired differentiation and the lack of transcriptional lineage priming in mutant cEpiSCs will be further deciphered in the following Chapter V and VI.
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