ZNF410 Uniquely Activates the NuRD Component CHD4 to Silence Fetal Hemoglobin Expression.

Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA-binding protein that in human erythroid cells directly activates only a single gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplantation settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels.. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells, and its perturbation might offer new opportunities for treatment of hemoglobinopathies.

Activation of γ-globin expression by hypoxia-inducible factor 1α.

Around birth, globin expression in human red blood cells (RBCs) shifts from γ-globin to ß-globin, which results in fetal haemoglobin (HbF, α2γ2) being gradually replaced by adult haemoglobin (HbA, α2ß2)1. This process has motivated the development of innovative approaches to treat sickle cell disease and ß-thalassaemia by increasing HbF levels in postnatal RBCs2. Here we provide therapeutically relevant insights into globin gene switching obtained through a CRISPR-Cas9 screen for ubiquitin-proteasome components that regulate HbF expression. In RBC precursors, depletion of the von Hippel-Lindau (VHL) E3 ubiquitin ligase stabilized its ubiquitination target, hypoxia-inducible factor 1α (HIF1α)3,4, to induce γ-globin gene transcription. Mechanistically, HIF1α-HIF1ß heterodimers bound cognate DNA elements in BGLT3, a long noncoding RNA gene located 2.7 kb downstream of the tandem γ-globin genes HBG1 and HBG2. This was followed by the recruitment of transcriptional activators, chromatin opening and increased long-range interactions between the γ-globin genes and their upstream enhancer. Similar induction of HbF occurred with hypoxia or with inhibition of prolyl hydroxylase domain enzymes that target HIF1α for ubiquitination by the VHL E3 ubiquitin ligase. Our findings link globin gene regulation with canonical hypoxia adaptation, provide a mechanism for HbF induction during stress erythropoiesis and suggest a new therapeutic approach for ß-haemoglobinopathies.

Disrupting the adult globin promoter alleviates promoter competition and reactivates fetal globin gene expression.

The benign condition hereditary persistence of fetal hemoglobin (HPFH) is known to ameliorate symptoms of co-inherited ß-hemoglobinopathies, such as sickle cell disease and ß-thalassemia. The condition is sometimes associated with point mutations in the fetal globin promoters that disrupt the binding of the repressors BCL11A or ZBTB7A/LRF, which have been extensively studied. HPFH is also associated with a range of deletions within the ß-globin locus that all reside downstream of the fetal HBG2 gene. These deletional forms of HPFH are poorly understood and are the focus of this study. Numerous different mechanisms have been proposed to explain how downstream deletions can boost the expression of the fetal globin genes, including the deletion of silencer elements, of genes encoding noncoding RNA, and bringing downstream enhancer elements into proximity with the fetal globin gene promoters. Here we systematically analyze the deletions associated with both HPFH and a related condition known as δß-thalassemia and propose a unifying mechanism. In all cases where fetal globin is upregulated, the proximal adult ß-globin (HBB) promoter is deleted. We use clustered regularly interspaced short palindromic repeats-mediated gene editing to delete or disrupt elements within the promoter and find that virtually all mutations that reduce ΗΒΒ promoter activity result in elevated fetal globin expression. These results fit with previous models where the fetal and adult globin genes compete for the distal locus control region and suggest that targeting the ΗΒΒ promoter might be explored to elevate fetal globin and reduce sickle globin expression as a treatment of ß-hemoglobinopathies.

Dynamic CTCF binding directly mediates interactions among cis-regulatory elements essential for hematopoiesis.

While constitutive CCCTC-binding factor (CTCF)-binding sites are needed to maintain relatively invariant chromatin structures, such as topologically associating domains, the precise roles of CTCF to control cell-type-specific transcriptional regulation remain poorly explored. We examined CTCF occupancy in different types of primary blood cells derived from the same donor to elucidate a new role for CTCF in gene regulation during blood cell development. We identified dynamic, cell-type-specific binding sites for CTCF that colocalize with lineage-specific transcription factors. These dynamic sites are enriched for single-nucleotide polymorphisms that are associated with blood cell traits in different linages, and they coincide with the key regulatory elements governing hematopoiesis. CRISPR-Cas9-based perturbation experiments demonstrated that these dynamic CTCF-binding sites play a critical role in red blood cell development. Furthermore, precise deletion of CTCF-binding motifs in dynamic sites abolished interactions of erythroid genes, such as RBM38, with their associated enhancers and led to abnormal erythropoiesis. These results suggest a novel, cell-type-specific function for CTCF in which it may serve to facilitate interaction of distal regulatory emblements with target promoters. Our study of the dynamic, cell-type-specific binding and function of CTCF provides new insights into transcriptional regulation during hematopoiesis.

FBXO11-mediated proteolysis of BAHD1 relieves PRC2-dependent transcriptional repression in erythropoiesis.

The histone mark H3K27me3 and its reader/writer polycomb repressive complex 2 (PRC2) mediate widespread transcriptional repression in stem and progenitor cells. Mechanisms that regulate this activity are critical for hematopoietic development but are poorly understood. Here we show that the E3 ubiquitin ligase F-box only protein 11 (FBXO11) relieves PRC2-mediated repression during erythroid maturation by targeting its newly identified substrate bromo adjacent homology domain-containing 1 (BAHD1), an H3K27me3 reader that recruits transcriptional corepressors. Erythroblasts lacking FBXO11 are developmentally delayed, with reduced expression of maturation-associated genes, most of which harbor bivalent histone marks at their promoters. In FBXO11-/- erythroblasts, these gene promoters bind BAHD1 and fail to recruit the erythroid transcription factor GATA1. The BAHD1 complex interacts physically with PRC2, and depletion of either component restores FBXO11-deficient erythroid gene expression. Our studies identify BAHD1 as a novel effector of PRC2-mediated repression and reveal how a single E3 ubiquitin ligase eliminates PRC2 repression at many developmentally poised bivalent genes during erythropoiesis.

Acute depletion of CTCF directly affects MYC regulation through loss of enhancer-promoter looping.

Numerous pieces of evidence support the complex, 3D spatial organization of the genome dictates gene expression. CTCF is essential to define topologically associated domain boundaries and to facilitate the formation of insulated chromatin loop structures. To understand CTCF's direct role in global transcriptional regulation, we integrated the miniAID-mClover3 cassette to the endogenous CTCF locus in a human pediatric B-ALL cell line, SEM, and an immortal erythroid precursor cell line, HUDEP-2, to allow for acute depletion of CTCF protein by the auxin-inducible degron system. In SEM cells, CTCF loss notably disrupted intra-TAD loops and TAD integrity in concurrence with a reduction in CTCF-binding affinity, while showing no perturbation to nuclear compartment integrity. Strikingly, the overall effect of CTCF's loss on transcription was minimal. Whole transcriptome analysis showed hundreds of genes differentially expressed in CTCF-depleted cells, among which MYC and a number of MYC target genes were specifically downregulated. Mechanically, acute depletion of CTCF disrupted the direct interaction between the MYC promoter and its distal enhancer cluster residing ∼1.8 Mb downstream. Notably, MYC expression was not profoundly affected upon CTCF loss in HUDEP-2 cells suggesting that CTCF could play a B-ALL cell line specific role in maintaining MYC expression.

Overview of the roles of Sox2 in stem cell and development.

Sox2 is well known for its functions in embryonic stem (ES) cell pluripotency, maintenance, and self-renewal, and it is an essential factor in generating inducible pluripotent stem (iPS) cells. It also plays an important role in development and adult tissue homeostasis of different tissues, especially the central nervous system. Increasing evidence has shown that aging is a stemness-related process in which Sox2 is also implicated as a key player, especially in the neural system. These distinct roles that Sox2 plays involve delicate regulatory networks consisting of other master transcription factors, microRNAs and signaling pathways. Additionally, the expression level of Sox2 can also be modulated transcriptionally, translationally or post-translationally. Here we will mainly review the roles of Sox2 in stem cell related development, homeostasis maintenance, aging processes, and the underlying molecular mechanisms involved.

Lysophosphatidic acid accelerates lung fibrosis by inducing differentiation of mesenchymal stem cells into myofibroblasts.

Lung fibrosis is characterized by vascular leakage and myofibroblast recruitment, and both phenomena are mediated by lysophosphatidic acid (LPA) via its type-1 receptor (LPA1). Following lung damage, the accumulated myofibroblasts activate and secrete excessive extracellular matrix (ECM), and form fibrotic foci. Studies have shown that bone marrow-derived cells are an important source of myofibroblasts in the fibrotic organ. However, the type of cells in the bone marrow contributing predominantly to the myofibroblasts and the involvement of LPA-LPA1 signalling in this is yet unclear. Using a bleomycin-induced mouse lung-fibrosis model with an enhanced green fluorescent protein (EGFP) transgenic mouse bone marrow replacement, we first demonstrated that bone marrow derived-mesenchymal stem cells (BMSCs) migrated markedly to the bleomycin-injured lung. The migrated BMSC contributed significantly to α-smooth muscle actin (α-SMA)-positive myofibroblasts. By transplantation of GFP-labelled human BMSC (hBMSC) or EGFP transgenic mouse BMSC (mBMSC), we further showed that BMSC might be involved in lung fibrosis in severe combined immune deficiency (SCID)/Beige mice induced by bleomycin. In addition, using quantitative-RT-PCR, western blot, Sircol collagen assay and migration assay, we determined the underlying mechanism was LPA-induced BMSC differentiation into myofibroblast and the secretion of ECM via LPA1. By employing a novel LPA1 antagonist, Antalpa1, we then showed that Antalpa1 could attenuate lung fibrosis by inhibiting both BMSC differentiation into myofibroblast and the secretion of ECM. Collectively, the above findings not only further validate LPA1 as a drug target in the treatment of pulmonary fibrosis but also elucidate a novel pathway in which BMSCs contribute to the pathologic process.

High-fat diet induces early-onset diabetes in heterozygous Pax6 mutant mice.

BACKGROUND: Type 2 diabetes is caused by interactions between genetic and environmental factors. Our previous studies reported that paired box 6 mutation heterozygosity (Pax6(m/+)) led to defective proinsulin processing and subsequent abnormal glucose metabolism in mice at 6 months of age. However, high-fat diet exposure could be an important incentive for diabetes development. In this study, we aimed to develop a novel diabetic model imitating human type 2 diabetes by exposing Pax6(m/+) mice to high-fat diet and to explore the underlying mechanism of diabetes in this model. METHODS: Over 300 Pax6(m/+) and wild-type male weanling mice were randomly divided into two groups and were fed an high-fat diet or chow diet for 6-10 weeks. Blood glucose and glucose tolerance levels were monitored during this period. Body weights, visceral adipose weights, blood lipid profiles and insulin sensitivity (determined with an insulin tolerance test) were used to evaluate obesity and insulin resistance. Proinsulin processing and insulin secretion levels were used to evaluate pancreatic ß cell function. RESULTS: After 6 weeks of high-fat diet exposure, only the Pax6(m/+) mice showed dramatic postloading hyperglycaemia. These mice exhibited significant high-fat diet-induced visceral obesity and insulin resistance and displayed defective prohormone convertase 1/3 production, an increased proinsulin:total insulin ratio and impaired early-phase insulin secretion, because of the Pax6 mutation. Hyperglycaemia worsened progressively over time with the high-fat diet, and most Pax6(m/+) mice on high-fat diet developed diabetes or impaired glucose tolerance after 10 weeks. Furthermore, high-fat diet withdrawal partly improved blood glucose levels in the diabetic mice. CONCLUSIONS: By combining the Pax6(m/+) genetic background with an high-fat diet environment, we developed a novel diabetic model to mimic human type 2 diabetes. This model is characterized by impaired insulin secretion, caused by the Pax6 mutation, and high-fat diet-induced insulin resistance and therefore provides an ideal tool for research on type 2 diabetes pathogenesis and therapies.

Sox2 protects neural stem cells from apoptosis via up-regulating survivin expression.

The transcription factor Sox2 [SRY (sex-determining region Y)-box 2] is essential for the regulation of self-renewal and homoeostasis of NSCs (neural stem cells) during brain development. However, the downstream targets of Sox2 and its underlying molecular mechanism are largely unknown. In the present study, we found that Sox2 directly up-regulates the expression of survivin, which inhibits the mitochondria-dependent apoptotic pathway in NSCs. Although overexpression of Sox2 elevates survivin expression, knockdown of Sox2 results in a decrease in survivin expression, thereby initiating the mitochondria-dependent apoptosis related to caspase 9 activation. Furthermore, cell apoptosis owing to knockdown of Sox2 can be rescued by ectopically expressing survivin in NSCs as well as in the mouse brain, as demonstrated by an in utero-injection approach. In short, we have found a novel Sox2/survivin pathway that regulates NSC survival and homoeostasis, thus revealing a new mechanism of brain development, neurological degeneration and such aging-related disorders.

PDGF mediates derivation of human embryonic germ cells.

Human embryonic germ cells (hEGCs) are a valuable and underutilized source of pluripotent stem cells. Unlike embryonic stem cells, which have been extensively studied, little is known about the factors that regulate hEGC derivation and maintenance. This study demonstrates for the first time a central role for selective activation of PDGFR signaling in the derivation and maintenance of pluripotency in hEGCs. In the study, hEGCs were found to express PDGF receptor α at high levels compared to human embryonic stem cells (hESCs). PDGF significantly improved formation of alkaline phosphatase (AP) positive hEGC colonies. We subsequently determined that PDGF activates the phosphatidylinositol-3-kinase (PI3K) pathway as phosphorylation of AKT was up-regulated in response to PDGF. Furthermore, inhibition of PI3K signaling using small molecular inhibitor LY294002 led to significantly decreased AP positive hEGC colony formation whereas inhibition of MAPK pathway using U0126 had a negligible effect. We established a primary mechanism for PDGF mediated derivation and maintenance of hEGCs by demonstrating that OCT4 was upregulated and PTEN was suppressed in a dose dependent manner in response to PDGF.

ETV6 represses inflammatory response genes and regulates HSPC function during stress hematopoiesis in mice.

ETS variant 6 (ETV6) encodes a transcriptional repressor expressed in hematopoietic stem and progenitor cells (HSPCs), where it is required for adult hematopoiesis. Heterozygous pathogenic germline ETV6 variants are associated with thrombocytopenia 5 (T5), a poorly understood genetic condition resulting in thrombocytopenia and predisposition to hematologic malignancies. To elucidate how germline ETV6 variants affect HSPCs and contribute to disease, we generated a mouse model harboring an Etv6R355X loss-of-function variant, equivalent to the T5-associated variant ETV6R359X. Under homeostatic conditions, all HSPC subpopulations are present in the bone marrow (BM) of Etv6R355X/+ mice; however, these animals display shifts in the proportions and/or numbers of progenitor subtypes. To examine whether the Etv6R355X/+ mutation affects HSPC function, we performed serial competitive transplantation and observed that Etv6R355X/+ lineage-sca1+cKit+ (LSK) cells exhibit impaired reconstitution, with near complete failure to repopulate irradiated recipients by the tertiary transplant. Mechanistic studies incorporating cleavage under target and release under nuclease assay, assay for transposase accessible chromatin sequencing, and high-throughput chromosome conformation capture identify ETV6 binding at inflammatory gene loci, including multiple genes within the tumor necrosis factor (TNF) signaling pathway in ETV6-sufficient mouse and human HSPCs. Furthermore, single-cell RNA sequencing of BM cells isolated after transplantation reveals upregulation of inflammatory genes in Etv6R355X/+ progenitors when compared to Etv6+/+ counterparts. Corroborating these findings, Etv6R355X/+ HSPCs produce significantly more TNF than Etv6+/+ cells post-transplantation. We conclude that ETV6 is required to repress inflammatory gene expression in HSPCs under conditions of hematopoietic stress, and this mechanism may be critical to sustain HSPC function.

Potent and uniform fetal hemoglobin induction via base editing.

Inducing fetal hemoglobin (HbF) in red blood cells can alleviate ß-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors. The most potent modification was adenine base editor generation of γ-globin -175A>G. Homozygous -175A>G edited erythroid colonies expressed 81 ± 7% HbF versus 17 ± 11% in unedited controls, whereas HbF levels were lower and more variable for two Cas9 strategies targeting a BCL11A binding motif in the γ-globin promoter or a BCL11A erythroid enhancer. The -175A>G base edit also induced HbF more potently than a Cas9 approach in red blood cells generated after transplantation of CD34+ hematopoietic stem and progenitor cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 can cause unexpected phenotypic variation that can be circumvented by base editing.

SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia.

Acute myeloid leukemia (AML) cells rely on phospho-signaling pathways to gain unlimited proliferation potential. Here, we use domain-focused CRISPR screening and identify the nuclear phosphatase SCP4 as a dependency in AML, yet this enzyme is dispensable in normal hematopoietic progenitor cells. Using CRISPR exon scanning and gene complementation assays, we show that the catalytic function of SCP4 is essential in AML. Through mass spectrometry analysis of affinity-purified complexes, we identify the kinase paralogs STK35 and PDIK1L as binding partners and substrates of the SCP4 phosphatase domain. We show that STK35 and PDIK1L function catalytically and redundantly in the same pathway as SCP4 to maintain AML proliferation and to support amino acid biosynthesis and transport. We provide evidence that SCP4 regulates STK35/PDIK1L through two distinct mechanisms: catalytic removal of inhibitory phosphorylation and by promoting kinase stability. Our findings reveal a phosphatase-kinase signaling complex that supports the pathogenesis of AML.

Dual function NFI factors control fetal hemoglobin silencing in adult erythroid cells.

The mechanisms by which the fetal-type ß-globin-like genes HBG1 and HBG2 are silenced in adult erythroid precursor cells remain a fundamental question in human biology and have therapeutic relevance to sickle cell disease and ß-thalassemia. Here, we identify via a CRISPR-Cas9 genetic screen two members of the NFI transcription factor family-NFIA and NFIX-as HBG1/2 repressors. NFIA and NFIX are expressed at elevated levels in adult erythroid cells compared with fetal cells, and function cooperatively to repress HBG1/2 in cultured cells and in human-to-mouse xenotransplants. Genomic profiling, genome editing and DNA binding assays demonstrate that the potent concerted activity of NFIA and NFIX is explained in part by their ability to stimulate the expression of BCL11A, a known silencer of the HBG1/2 genes, and in part by directly repressing the HBG1/2 genes. Thus, NFI factors emerge as versatile regulators of the fetal-to-adult switch in ß-globin production.

The EGF receptor-sox2-EGF receptor feedback loop positively regulates the self-renewal of neural precursor cells.

The transcriptional factor Sox2 and epidermal growth factor receptor (Egfr)-mediated signaling are both required for self-renewal of neural precursor cells (NPCs). However, the mechanism by which these factors coordinately regulate this process is largely unknown. Here we show that Egfr-mediated signaling promotes Sox2 expression, which in turn binds to the Egfr promoter and directly upregulates Egfr expression. Knockdown of Sox2 by RNA interference downregulates Egfr expression and attenuates colony formation of NPCs, whereas overexpression of Sox2 elevates Egfr expression and promotes NPC self-renewal. Moreover, the effect of Sox2 on NPC self-renewal is completely inhibited by AG1478, a specific inhibitor for Egfr; it is also inhibited by LY294002 and U0126, selective antagonists for phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (Erk1/2), respectively. Collectively, we conclude that NPC self-renewal is enhanced through a novel cellular feedback loop with mutual regulation of Egfr and Sox2.

Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin.

Hereditary persistence of fetal hemoglobin (HPFH) ameliorates ß-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to ß-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of nuclear transcription factor Y (NF-Y) to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.

Single-nucleotide-level mapping of DNA regulatory elements that control fetal hemoglobin expression.

Pinpointing functional noncoding DNA sequences and defining their contributions to health-related traits is a major challenge for modern genetics. We developed a high-throughput framework to map noncoding DNA functions with single-nucleotide resolution in four loci that control erythroid fetal hemoglobin (HbF) expression, a genetically determined trait that modifies sickle cell disease (SCD) phenotypes. Specifically, we used the adenine base editor ABEmax to introduce 10,156 separate A•T to G•C conversions in 307 predicted regulatory elements and quantified the effects on erythroid HbF expression. We identified numerous regulatory elements, defined their epigenomic structures and linked them to low-frequency variants associated with HbF expression in an SCD cohort. Targeting a newly discovered γ-globin gene repressor element in SCD donor CD34+ hematopoietic progenitors raised HbF levels in the erythroid progeny, inhibiting hypoxia-induced sickling. Our findings reveal previously unappreciated genetic complexities of HbF regulation and provide potentially therapeutic insights into SCD.

Application of amphiphilic fluorophore-derived nanoparticles to provide contrast to human embryonic stem cells without affecting their pluripotency and to monitor their differentiation into neuron-like cells.

Fluorogenic labeling is a potential technique in biology that allows for direct detection and tracking of cells undergoing various biological processes. Compared to traditional genetic modification approaches, labeling cells with nanoparticles has advantages, especially for the additional safety they provide by avoiding genomic integration. However, it remains a challenge to determine whether nanoparticles interfere with cell traits and provide long-lasting signals in living cells. We employed an amphiphilic fluorophore-derived nanoparticle (denoted by TPE-11) bearing a tetraphenylethene (TPE) moiety and two ionic heads; this nanoparticle has an aggregation-induced emission (AIE) effect and the ability to self-assemble. TPE-11 exhibited the property of higher or longer fluorescence intensities in cell imaging than the other two nanomaterials under the same conditions. We used this nanomaterial to label human embryonic stem (hES) cells and monitor their differentiation. Treatment with low concentrations of TPE-11 (8.0 µg/mL) resulted in high-intensity labeling of hES cells, and immunostaining analysis and teratoma formation assays showed that at this concentration, their pluripotency remained unaltered. TPE-11 nanoparticles allowed for long-term monitoring of hES cell differentiation into neuron-like cells; remarkably, strong nanoparticle signals were detected throughout the nearly 40-day differentiation process. Thus, these results demonstrate that the TPE-11 nanoparticle has excellent biocompatibility for hES cells and is a potential fluorogen for labeling and tracking the differentiation of human pluripotent stem cells. STATEMENT OF SIGNIFICANCE: This study uses a nanoparticle-based approach to label human embryonic stem (hES) cells and monitor their differentiation. hES cells are distinguished by two distinctive properties: the state of their pluripotency and the potential to differentiate into various cell types. Thus, these cells will be useful as a source of cells for transplantation or tissue engineering applications. We noticed the effect of aggregation-induced emission, and the ability to self-assemble could enhance the persistence of signals. Treatment with low concentrations of TPE-11 nanoparticles showed high-intensity labeling of hES cells, and immunostaining analysis and teratoma formation assays showed that at this concentration, their pluripotency remained unaltered. Additionally, these nanoparticles allowed for long-term monitoring of hES cell differentiation into neuron-like cells lasting for 40 days.

An Eye Organoid Approach Identifies Six3 Suppression of R-spondin 2 as a Critical Step in Mouse Neuroretina Differentiation.

Recent advances in self-organizing, 3-dimensional tissue cultures of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provided an in vitro model that recapitulates many aspects of the in vivo developmental steps. Using Rax-GFP-expressing ESCs, newly generated Six3-/- iPSCs, and conditional null Six3delta/f;Rax-Cre ESCs, we identified Six3 repression of R-spondin 2 (Rspo2) as a required step during optic vesicle morphogenesis and neuroretina differentiation. We validated these results in vivo by showing that transient ectopic expression of Rspo2 in the anterior neural plate of transgenic mouse embryos was sufficient to inhibit neuroretina differentiation. Additionally, using a chimeric eye organoid assay, we determined that Six3 null cells exert a non-cell-autonomous repressive effect during optic vesicle formation and neuroretina differentiation. Our results further validate the organoid culture system as a reliable and fast alternative to identify and evaluate genes involved in eye morphogenesis and neuroretina differentiation in vivo.