The maintenance of pluripotency requires coordinated expression of a set of essential genes. Using our recently established haploid human pluripotent stem cells (hPSCs), we generated a genome-wide loss-of-function library targeting 18,166 protein-coding genes to define the essential genes in hPSCs. With this we could allude to an intrinsic bias of essentiality across cellular compartments, uncover two opposing roles for tumour suppressor genes and link autosomal-recessive disorders with growth-retardation phenotypes to early embryogenesis. hPSC-enriched essential genes mainly encode transcription factors and proteins related to cell-cycle and DNA-repair, revealing that a quarter of the nuclear factors are essential for normal growth. Our screen also led to the identification of growth-restricting genes whose loss of function provides a growth advantage to hPSCs, highlighting the role of the P53-mTOR pathway in this context. Overall, we have constructed an atlas of essential and growth-restricting genes in hPSCs, revealing key aspects of cellular essentiality and providing a reference for future studies on human pluripotency.

Duchenne muscular dystrophy (DMD) is caused by mutations in dystrophin and the subsequent disruption of the dystrophin-associated protein complex (DAPC). Utrophin is a dystrophin homolog expressed at high levels in developing muscle that is an attractive target for DMD therapy. Here we show that the extracellular matrix protein biglycan regulates utrophin expression in immature muscle and that recombinant human biglycan (rhBGN) increases utrophin expression in cultured myotubes. Systemically delivered rhBGN upregulates utrophin at the sarcolemma and reduces muscle pathology in the mdx mouse model of DMD. RhBGN treatment also improves muscle function as judged by reduced susceptibility to eccentric contraction-induced injury. Utrophin is required for the rhBGN therapeutic effect. Several lines of evidence indicate that biglycan acts by recruiting utrophin protein to the muscle membrane. RhBGN is well tolerated in animals dosed for as long as 3 months. We propose that rhBGN could be a therapy for DMD.biotherapeutics | protein therapeutics D uchenne muscular dystrophy (DMD) is a hereditary disease that affects ∼1:3,500 boys, the majority of whom will die by their midtwenties (1). DMD is caused by mutations in dystrophin that result in the faulty assembly and function of an ensemble of structural and signaling molecules at the muscle cell surface termed the dystrophin-associated protein complex (DAPC) (2-4). There are currently no treatments that target the primary pathology of DMD.One attractive therapeutic approach for DMD is the stabilization of the muscle cell membrane through up-regulation of utrophin, a dystrophin homolog. Transgenic overexpression of utrophin rescues dystrophic pathology and restores function in the dystrophin-deficient mdx mouse (5-7). In mature muscle, utrophin expression is restricted to the neuromuscular and myotendinous junctions. However, utrophin is expressed over the entire myofiber in developing and regenerating muscle (8-10). These observations raise the possibility that marshalling pathways that normally regulate utrophin expression in developing muscle could be a productive approach for developing DMD treatments.The extracellular matrix protein biglycan plays an important role in developing muscle. In both humans and mice, biglycan is most highly expressed in immature and regenerating muscle (11,12). Biglycan is a component of the DAPC, where it binds to α-dystroglycan (13) and α-and γ-sarcoglycan (14). Biglycan regulates the expression of the sarcoglycans as well as dystrobrevins, syntrophins, and nNOS, particularly in immature muscle. Finally, biglycan is important for timely muscle regeneration (11).Locally delivered recombinant human biglycan (rhBGN) incorporates into the extracellular matrix of bgn −/o muscle where it persists for at least 2 wk and rescues the expression of several DAPC components (15). These results raise the possibility that rhBGN might enhance function in muscle that lacks dystrophin. Here we show that utrophin is down-regulated in immature biglycan null (...

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Bone morphogenetic proteins (BMPs) function in most tissues but have cell type-specific effects. Given the relatively small number of BMP receptors, this exquisite signaling specificity requires additional molecules to regulate this pathway’s output. The receptor tyrosine kinase MuSK (muscle-specific kinase) is critical for neuromuscular junction formation and maintenance. Here, we show that MuSK also promotes BMP signaling in muscle cells. MuSK bound to BMP4 and related BMPs with low nanomolar affinity in vitro and to the type I BMP receptors ALK3 and ALK6 in a ligand-independent manner both in vitro and in cultured myotubes. High-affinity binding to BMPs required the third, alternatively spliced MuSK immunoglobulin-like domain. In myoblasts, endogenous MuSK promoted BMP4-dependent phosphorylation of SMADs and transcription of Id1, which encodes a transcription factor involved in muscle differentiation. Gene expression profiling showed that MuSK was required for the BMP4-induced expression of a subset of genes in myoblasts, including regulator of G protein signaling 4 (Rgs4). In myotubes, MuSK enhanced the BMP4-induced expression of a distinct set of genes, including transcripts characteristic of slow muscle. MuSK-mediated stimulation of BMP signaling required type I BMP receptor activity but was independent of MuSK tyrosine kinase activity. MuSK-dependent expression of Rgs4 resulted in the inhibition of Ca2+ signaling induced by the muscarinic acetylcholine receptor in myoblasts. These findings establish that MuSK has dual roles in muscle cells, acting both as a tyrosine kinase-dependent synaptic organizing molecule and as a BMP co-receptor that shapes BMP transcriptional output and cholinergic signaling.

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Highlights d CRISPR screens reveal genes essential for germ layerdifferentiation of human ESCs d Several classes of developmental diseases exhibit early differentiation phenotypes d Germ layer-specific essential transcription factors manifest a functional hierarchy d Exit from pluripotency is orchestrated by proteins within the ER-Golgi network

Recent advances in the generation of haploid embryonic stem cells (ESCs), capable of self-renewal and differentiation, have laid the groundwork for numerous biomedical applications in developmental biology and reproductive medicine. When combined with the power of genetic screening, haploid human ESCs could advance cancer research, regenerative medicine, and disease modeling.

In mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-of-origin. Yet, it is largely unknown how imprinted DMRs are maintained in human embryos despite global DNA demethylation following fertilization. Here, we explored the mechanisms involved in imprinting regulation by employing human parthenogenetic embryonic stem cells (hpESCs), which lack paternal alleles. We show that although global loss of DNA methylation in hpESCs affects most imprinted DMRs, many paternally-expressed genes (PEGs) remain repressed. To search for factors regulating PEGs, we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs. This revealed ATF7IP as an essential repressor of a set of PEGs, which we further show is also required for silencing sperm-specific genes. Our study reinforces an important role for histone modifications in regulating imprinted genes and suggests a link between parental imprinting and germ cell identity.