An O-GlcNAc transferase pathogenic variant linked to intellectual disability affects pluripotent stem cell self-renewal.

O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential enzyme that modifies proteins with O-GlcNAc. Inborn OGT genetic variants were recently shown to mediate a novel type of congenital disorder of glycosylation (OGT-CDG), which is characterised by X-linked intellectual disability (XLID) and developmental delay. Here, we report an OGTC921Y variant that co-segregates with XLID and epileptic seizures, and results in loss of catalytic activity.

An ERK5-KLF2 signalling module regulates early embryonic gene expression and telomere rejuvenation in stem cells.

The ERK5 MAP kinase signalling pathway drives transcription of naïve pluripotency genes in mouse Embryonic Stem Cells (mESCs). However, how ERK5 impacts on other aspects of mESC biology has not been investigated. Here, we employ quantitative proteomic profiling to identify proteins whose expression is regulated by the ERK5 pathway in mESCs.

An intellectual disability syndrome with single-nucleotide variants in O-GlcNAc transferase.

Intellectual disability (ID) is a neurodevelopmental condition that affects ~1% of the world population. In total 5-10% of ID cases are due to variants in genes located on the X chromosome. Recently, variants in OGT have been shown to co-segregate with X-linked intellectual disability (XLID) in multiple families.

A missense mutation in the catalytic domain of O-GlcNAc transferase links perturbations in protein O-GlcNAcylation to X-linked intellectual disability.

X-linked intellectual disabilities (XLID) are common developmental disorders. The enzyme O-GlcNAc transferase encoded by OGT, a recently discovered XLID gene, attaches O-GlcNAc to nuclear and cytoplasmic proteins. As few missense mutations have been described, it is unclear what the aetiology of the patient phenotypes is.

Catalytic deficiency of O-GlcNAc transferase leads to X-linked intellectual disability.

O-GlcNAc transferase (OGT) is an X-linked gene product that is essential for normal development of the vertebrate embryo. It catalyses the O-GlcNAc posttranslational modification of nucleocytoplasmic proteins and proteolytic maturation of the transcriptional coregulator Host cell factor 1 (HCF1). Recent studies have suggested that conservative missense mutations distal to the OGT catalytic domain lead to X-linked intellectual disability in boys, but it is not clear if this is through changes in the O-GlcNAc proteome, loss of protein-protein interactions, or misprocessing of HCF1.

Neural differentiation of mouse embryonic stem cells in serum-free monolayer culture.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days.

Elevated O-GlcNAc levels activate epigenetically repressed genes and delay mouse ESC differentiation without affecting naïve to primed cell transition.

The differentiation of mouse embryonic stem cells (ESCs) is controlled by the interaction of multiple signaling pathways, typically mediated by post-translational protein modifications. The addition of O-linked N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of nuclear and cytoplasmic proteins is one such modification (O-GlcNAcylation), whose function in ESCs is only now beginning to be elucidated. Here, we demonstrate that the specific inhibition of O-GlcNAc hydrolase (Oga) causes increased levels of protein O-GlcNAcylation and impairs differentiation of mouse ESCs both in serum-free monolayer and in embryoid bodies (EBs).

Specific glycosaminoglycans modulate neural specification of mouse embryonic stem cells.

Mouse embryonic stem (mES) cells express a low sulfated form of heparan sulfate (HS). HS chains displayed by ES cells and their progeny become more complex and more sulfated during progression from pluripotency to neuroectodermal precursors. Sulfated epitopes are important for recognition and binding of a variety of ligands including members of the fibroblast growth factor (FGF) family.

Retinoic acid orchestrates fibroblast growth factor signalling to drive embryonic stem cell differentiation.

Embryonic stem (ES) cells fluctuate between self-renewal and the threshold of differentiation. Signalling via the fibroblast growth factor (Fgf)/Erk pathway is required to progress from this dynamic state and promote mouse ES cell differentiation. Retinoic acid also induces differentiation in many cellular contexts, but its mechanism of action in relation to Fgf/Erk signalling in ES cells is poorly understood.

Negative-feedback regulation of FGF signalling by DUSP6/MKP-3 is driven by ERK1/2 and mediated by Ets factor binding to a conserved site within the DUSP6/MKP-3 gene promoter.

DUSP6 (dual-specificity phosphatase 6), also known as MKP-3 [MAPK (mitogen-activated protein kinase) phosphatase-3] specifically inactivates ERK1/2 (extracellular-signal-regulated kinase 1/2) in vitro and in vivo. DUSP6/MKP-3 is inducible by FGF (fibroblast growth factor) signalling and acts as a negative regulator of ERK activity in key and discrete signalling centres that direct outgrowth and patterning in early vertebrate embryos. However, the molecular mechanism by which FGFs induce DUSP6/MKP-3 expression and hence help to set ERK1/2 signalling levels is unknown.

A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification.

Neural tissue formation is induced by growth factors that activate networks of signal transduction cascades that ultimately lead to the expression of early neural genes, including transcription factors of the SoxB family. Here, we report that fibroblast growth factor (FGF)-induced Erk1/2 (Mapk3 and Mapk1, respectively) mitogen-activated protein kinase (MAPK), but not phosphatidylinositol 3'-OH kinase (PI3K, Pik3r1), signalling is required for neural specification in mouse embryonic stem (ES) cells and in the chick embryo. Further, blocking Erk1/2 inhibits the onset of key SoxB genes in both mouse ES cells (Sox1) and chick embryos (Sox2 and Sox3) and, in both contexts, Erk1/2 signalling is required during only a narrow time window, as neural specification takes place.

Essential alterations of heparan sulfate during the differentiation of embryonic stem cells to Sox1-enhanced green fluorescent protein-expressing neural progenitor cells.

Embryonic stem (ES) cells can be cultured in conditions that either maintain pluripotency or allow differentiation to the three embryonic germ layers. Heparan sulfate (HS), a highly polymorphic glycosaminoglycan, is a critical cell surface coreceptor in embryogenesis, and in this paper we describe its structural transition from an unusually low-sulfated variant in ES cells to a more highly sulfated form in fluorescence-activated cell sorting-purified neural progenitor cells. The characteristic domain structure of HS was retained during this transformation.

Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice.

The transcription factor Sox1 is the earliest and most specific known marker for mammalian neural progenitors. During fetal development, Sox1 is expressed by proliferating progenitor cells throughout the central nervous system and in no tissue but the lens. We generated a reporter mouse line in which egfp is inserted into the Sox1 locus.

Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture.

Mouse embryonic stem (ES) cells are competent for production of all fetal and adult cell types. However, the utility of ES cells as a developmental model or as a source of defined cell populations for pharmaceutical screening or transplantation is compromised because their differentiation in vitro is poorly controlled. Specification of primary lineages is not understood and consequently differentiation protocols are empirical, yielding variable and heterogeneous outcomes.