A Nuclear Mechanoresponse to Morphogenesis Drives Acquisition of Neuroectoderm Lineage Competence During Pluripotent Cell Differentiation

Abstract

During development, cell differentiation involves morphogenetic transformations that shape and organize tissues. Emerging evidence suggests that the nucleus is mechanosensitive and that the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex can transduce morphogenesis-associated, actomyosin-generated cellular forces to the nucleus and influence cell state. Nuclear mechanosensitivity can result in changes a cell’s molecular-epigenetic state and regulate gene expression during differentiation, thereby influencing cell fate. During implantation, pluripotent cells within the embryo undergo an apical constriction and epithelialization morphogenesis in parallel with a cell state transition to prepare for multilineage differentiation. I therefore hypothesized that pluripotency and subsequent cell fate specification involves nuclear mechanosensitivity to morphogenesis-associated forces. To test this, I investigated the role of actomyosin contractility and the LINC complex in a model of exit from naïve pluripotency and neuroectoderm lineage differentiation with mouse embryonic stem cells (mESCs). Through a series of live and fixed cell fluorescence imaging experiments, I demonstrated that in adherent culture, pluripotent cells can recapitulate aspects of apical constriction morphogenesis during initial exit from naïve pluripotency. Apical constriction was marked by an apical accumulation of actomyosin that colocalized with phosphorylated myosin light chain. Next, pharmacological inhibition of actomyosin contractility resulted in normal progression of pluripotency but impaired commitment to the neuroectoderm lineage, marked by reduced expression of master lineage transcription factor, SOX1. Next, I sought to determine if actomyosin contractility control of neuroectoderm lineage specification was dependent on the LINC complex. To this end, mESCs with inducible dominant negative disruption of the LINC complex, or overexpression of ectopic elements of the nuclear lamina, were validated and tested. Disruption of the LINC complex also had no detectable effect on the progression of pluripotency but resulted in impaired commitment to the neuroectoderm lineage. Emerin is a mechanosensitive, LINC-interacting, nuclear envelope protein that can regulate histone-modifying enzyme activity. Emerin knock-down also resulted in impaired neuroectoderm differentiation. Interestingly, perturbations of contractility or LINC only led to an impaired differentiation phenotype if induced before the window of apical constriction morphogenesis, and not if induced shortly after. The time dependence for nuclear envelope perturbations to produce a differentiation phenotype suggested a molecular-epigenetic basis of Sox1 regulation, driven by a transient window of morphogenesis-associated actomyosin contractility and LINC complex function. Supporting this, analysis of histone modifications associated with gene repression and regulation revealed striking contractility and LINC-dependent changes during apical constriction. Most notable was a loss of H3K9me3 at the nuclear periphery, which marks dense and silent chromatin. There was also a contractility and LINC-dependent enrichment of emerin on the outer nuclear membrane during apical constriction. Furthermore, 3D DNA FISH revealed repositioning of Sox1 loci to the apical nuclear periphery during exit from naïve pluripotency, when apical constriction occurs. H3K27me3 is another repressive histone modification that is thought to poise genes for later activation and known to be specifically required to poise Sox1 for gene expression in differentiating mESCs. With ChIP-qPCR, I also demonstrated a LINC and actomyosin contractility dependent enrichment of H3K27me3 at the Sox1 promoter. Altogether, these data suggest that morphogenesis-associated cytoskeletal contractility and the LINC complex instruct chromatin changes, likely involving emerin and similar nuclear envelope proteins, that poise Sox1 for timely downstream expression. In short, my work demonstrates the role of a nuclear mechanoresponse in enabling acquisition of neuroectoderm lineage competence during exit from naïve pluripotency. This work demonstrates the advantages of an adherent model system which can be further exploited to understand the underlying mechanisms that drive apical constriction morphogenesis during pluripotent cell development. Further work will also focus on using genome-wide approaches to determine the subsets of genes that are responsive to the nuclear mechanoresponse to morphogenesis. There is also interest in further characterizing the mechanisms underlying contractility and LINC induced changes in histone modification distributions that result in regulation of lineage defining genes.