Oct4 Is Required ∼E7.5 for Proliferation in the Primitive Streak

<div><p>Oct4 is a widely recognized pluripotency factor as it maintains Embryonic Stem (ES) cells in a pluripotent state, and, <i>in vivo</i>, prevents the inner cell mass (ICM) in murine embryos from differentiating into trophectoderm. However, its function in somatic tissue after this developmental stage is not well characterized. Using a tamoxifen-inducible Cre recombinase and floxed alleles of Oct4, we investigated the effect of depleting Oct4 in mouse embryos between the pre-streak and headfold stages, ∼E6.0–E8.0, when Oct4 is found in dynamic patterns throughout the embryonic compartment of the mouse egg cylinder. We found that depletion of Oct4 ∼E7.5 resulted in a severe phenotype, comprised of craniorachischisis, random heart tube orientation, failed turning, defective somitogenesis and posterior truncation. Unlike in ES cells, depletion of the pluripotency factors Sox2 and Oct4 after E7.0 does not phenocopy, suggesting that ∼E7.5 Oct4 is required within a network that is altered relative to the pluripotency network. Oct4 is not required in extraembryonic tissue for these processes, but is required to maintain cell viability in the embryo and normal proliferation within the primitive streak. Impaired expansion of the primitive streak occurs coincident with Oct4 depletion ∼E7.5 and precedes deficient convergent extension which contributes to several aspects of the phenotype.</p></div

Decreased proliferation in the primitive streak occurs coincident with Oct4 depletion.

<p>The embryos depicted in panels ‘A–H’ were administered tamoxifen ∼E7.0 and ∼E7.5 and dissected 24 hrs ATA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003957#pgen.1003957.s012" target="_blank">Table S1A</a>C). Scale bars in ‘A–H’ are 50 µm. <b>A, B</b> Specification of Chordin in the node still occurs after Oct4 depletion <b>A</b> Oct4^f/f 24 hrs ATA. <b>B</b> Oct4^f/f;CreER^T2+/− 24 hrs ATA. <b>C, D</b> The expression domain of p-Smad1 is altered after Oct4 depletion. <b>C</b> Oct4^f/f 24 hrs ATA <b>D</b> Oct4^f/f;CreER^T2+/− 24 hrs ATA. <b>E,F</b> Distribution of apoptotic Caspase-3+ cells 24 hrs ATA. <b>E</b> Oct4^f/f<b>F</b> Oct4^f/f;CreER^T2+/−. <b>G,H</b> Distribution of Phospho-histone-3 (Ph3), which marks proliferating cells, 24 hrs ATA. Proliferation is significantly reduced in the primitive streak (bracketed by a white line) of Oct4^f/f;CreER^T2+/− embryos. (G) Oct4^f/f (H) Oct4^f/f;CreER^T2+/−. <b>I</b> Quantification of apoptotic frequency ±s.e.m. in Oct4^f/f and Oct4^f/f;CreER^T2+/− embryos (F_1,44 = 13.16, p<0.05 2-way ANOVA, *p<0.05, **p<0.01 Bonferroni posttest). <b>J</b> Quantification of proliferation frequency ±s.e.m. in Oct4^f/f and Oct4^f/f;CreER^T2+/− embryos 24 hrs ATA (F_1,68 = 3.28, p<0.05 2-way ANOVA, **p<0.01 Bonferroni posttest). Oct4 removal only effects proliferation significantly in the primitive streak. <b>K</b> Distribution of gene expression changes in Oct4^f/f;CreER^T2+/− embryos. Relative (Oct4^f/f;CreER^T2+/−/Oct4^f/f) transcript abundance ±s.e.m. was quantified 24 hrs ATA by QPCR (F_2,8 = 12.14, p<0.05 2-way ANOVA, ***p<0.001 Bonferroni posttest). Embryos were administered tamoxifen ∼E7.0 and ∼E7.5 and dissected 24 hrs ATA for the experiment in panel ‘K’ (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003957#pgen.1003957.s012" target="_blank">Table S1A</a>H).</p

Tetraploid chimeras indicate that Oct4 is not required extraembryonically ∼E7.5, while diploid chimeras indicate that Oct4^+/+ can compensate for Oct4^−/− cells embryonically.

<p>All embryos transferred to a surrogate and depicted or described in panels A–E were induced with tamoxifen ∼E6.0 and ∼E6.5 to compensate for the variability in developmental timing associated with transfer (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003957#pgen.1003957.s012" target="_blank">Table S1Q</a>,AB). Scale bars in panels A–D are 200 um. <b>A</b> A representative embryo from aggregation of Oct4^+/+ RFP ES cells with a tetraploid Oct4^f/f embryo. Oct4^f/f extraembryonic tissue yielded E9.5 chimeric embryos with no phenotype. <b>B</b> An Oct4^f/f;CreER^T2+/− E9.5 embryo with the Oct4^{COND MUT} phenotype. <b>C</b> Oct4 depletion in extraembryonic tissue is compatible with WT development. A representative chimera consisting of RFP+ Oct4^+/+ ES cell derived embryo and tetraploid Oct4^f/f;CreER^T2+/− extraembryonic tissue. The embryo has turned (compare panel ‘B’ where the tail is behind to panel ‘C’ where it is in front), undergone NTC and posterior extension (compare the lack of somites and short tail in panel ‘B’ to the somites and full-length tail in ‘C’). <b>D</b> The most severe embryonic defect observed in a diploid chimeras consisting of Oct4^+/+ and Oct4^f/f;CreER^T2+/− cells. The neural tube is open between closure points 1 and 2, indicated here with a black bracket. All Oct4^{COND MUT} features aside from cranial NTC defect, which is still present in 5/16 mosaic embryos, are rescued by Oct4^+/+ cells in these diploid chimeras (16/16). For example, this embryo has ‘turned’ such that it faces its tail and the posterior has extended normally. <b>E</b> Quantification of the genotypes and phenotypes of recovered chimeric embryos.</p

Pathway enrichment and confirmation of a subset of differentially expressed genes following Oct4 depletion.

<p>Litters depicted in ‘A,B’ were induced with tamoxifen ∼E7.0 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003957#pgen.1003957.s012" target="_blank">Table S1A</a>E). The FDR for reported enrichments in ‘A,B’ is <0.001, based on 1000 random permutations of annotated genes. <b>A</b> Unsupervised clustering of relative (Oct4^f/f;CreER^T2+/−/Oct4^f/f littermates) gene expression sub-setted for Oct4 binding targets following Oct4 depletion. Enrichment for the same pathways in the global differential expression set and subset directly targeted by Oct4 support the utility of sub-setting for Oct4 binding targets in identifying primary effects of Oct4 depletion and the relevance of these primary effects to the Oct4^{COND MUT} phenotype in that they appear amplified into effects on the overall gene expression profile (see blue script in panel ‘A’ and ‘B’ for these). The most enriched pathway is provided for each cluster, and an additional pathway provided (in black text) for the cluster where the most enriched pathway in the Oct4 target set did not translate to a global change. <b>B</b> Unsupervised clustering of global differential expression (same dataset as panel ‘A’): Oct4^f/f;CreER^T2+/−/Oct4^f/f. The most enriched pathway and binding factor are provided for each cluster (black text), while primary effects that translated to enriched effects in the global set are in blue text. <b>C</b> Confirmation of expression change for select genes by quantitative PCR in independent litters (Oct4^f/f;CreER^T2+/−/Oct4^f/f ±s.e.m.). Litters were induced with tamoxifen ∼E7.0 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003957#pgen.1003957.s012" target="_blank">Table S1A</a>G).</p

Gene expression profiling coincident with and following Oct4 depletion indicates that c-Myc, Smad1 and Oct4 targets are up-regulated while targets co-occupied by Oct4 and Sox2 are down-regulated.

<p><b>A</b> Combinations of TFs whose target sets were tested for enrichment amongst differentially expressed genes. <b>B,C</b> After the loss of Oct4 up-regulated genes are consistently enriched for targets of c-Myc, Smad1 and Oct4 while down-regulated genes are enriched for targets bound by both Oct4 and Sox2. Litters were induced with tamoxifen ∼E7.0 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003957#pgen.1003957.s012" target="_blank">Table S1A</a>E). The FDR for reported enrichments in ‘B,C’ is <0.001, based on 1000 random permutations of annotated genes. <b>B</b> TF binding enrichment amongst up-regulated genes using hypergeometric tests. <b>C</b> TF binding enrichment amongst down-regulated genes using hypergeometric tests.</p