Tbl1 promotes Wnt-ß-catenin signaling-induced degradation of the Tcf7l1 protein in mouse embryonic stem cells.

Activation of the Wnt-ß-catenin signaling pathway by CHIR99021, a specific inhibitor of GSK3ß, induces Tcf7l1 protein degradation, which facilitates the maintenance of an undifferentiated state in mouse embryonic stem cells (mESCs); however, the precise mechanism is still unclear. Here, we showed that the overexpression of transducin-ß-like protein 1 (Tbl1, also known as Tbl1x) or its family member Tblr1 (also known as Tbl1xr1) can decrease Tcf7l1 protein levels, whereas knockdown of each gene increases Tcf7l1 levels without affecting Tcf7l1 transcription. Interestingly, only Tbl1, and not Tblr1, interacts with Tcf7l1. Mechanistically, Tbl1 translocates from the cytoplasm into the nucleus in association with ß-catenin (CTNNB1) after the addition of CHIR99021 and functions as an adaptor to promote ubiquitylation of the Tcf7l1 protein. Functional assays further revealed that enforced expression of Tbl1 is capable of delaying mESC differentiation. In contrast, knockdown of Tbl1 attenuates the effect of CHIR99021 on Tcf7l1 protein stability and mESC self-renewal. Our results provide insight into the regulatory network of the Wnt-ß-catenin signaling pathway involved in promoting the maintenance of naïve pluripotency.

miR-10167-3p targets TCF7L1 to inhibit bovine adipocyte differentiation and promote bovine adipocyte proliferation.

MicroRNAs (miRNAs) are widely involved in various lipogenic processes, including adipocyte proliferation and differentiation, lipid droplet formation, and adipocyte-specific gene activation. The present study aimed to investigate the gene expression profiles of bovine preadipocytes under high miR-10167-3p expression using the RNA-seq technique and to verify the functions of its downstream target genes on the proliferation and differentiation of bovine preadipocytes. First, RNA-seq identified 573 differentially expressed genes (DEGs), of which 243 were downregulated and 330 were upregulated. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that 15.19% of the DEGs were enriched in pathways related to lipid metabolism. Meanwhile, dual-luciferase reporter gene assay verified the target-binding relationship between miR-10167-3p and TCF7L1. The function of TCF7L1 was assessed using several experiments in adipocytes with high TCF7L1 expression and RNA interference. The mRNA and protein expression of proliferation, differentiation, and apoptosis marker genes were detected using qPCR and western blot, respectively; lipid droplet synthesis was detected using oil red O, Nile red, and bodipy staining; adipocyte proliferation was detected by EdU; and apoptosis was detected using flow cytometry. The results revealed that TCF7L1 overexpression inhibited bovine preadipocyte differentiation and apoptosis and promoted their proliferation, with opposite results obtained with its RNA interference. These results may provide a reference for the subsequent investigation of the molecular mechanism of bovine fat deposition.

Characterization of a chromatin-associated TCF7L1 complex in human embryonic stem cells.

Human embryonic stem cells (hESCs) resemble the pluripotent epiblast cells found in the early postimplantation human embryo and represent the "primed" state of pluripotency. One factor that helps primed pluripotent cells retain pluripotency and prepare genes for differentiation is the transcription factor TCF7L1, a member of a small family of proteins known as T cell factors/Lymphoid enhancer factors (TCF/LEF) that act as downstream components of the WNT signaling pathway. Transcriptional output of the WNT pathway is regulated, in part, by the activity of TCF/LEFs in conjunction with another component of the WNT pathway, ß-CATENIN. Because TCF7L1 plays an important role in regulating pluripotency, we began to characterize the protein complex associated with TCF7L1 when bound to chromatin in hESCs using rapid immunoprecipitation of endogenous proteins (RIME).  Data are available via ProteomeXchange with identifier PXD047582. These data identify known and new partners of TCF7L1 on chromatin and provide novel insights into how TCF7L1 and pluripotency itself might be regulated.

TCF7L1 suppresses primitive streak gene expression to support human embryonic stem cell pluripotency.

Human embryonic stem cells (hESCs) are exquisitely sensitive to WNT ligands, which rapidly cause differentiation. Therefore, hESC self-renewal requires robust mechanisms to keep the cells in a WNT inactive but responsive state. How they achieve this is largely unknown. We explored the role of transcriptional regulators of WNT signaling, the TCF/LEFs. As in mouse ESCs, TCF7L1 is the predominant family member expressed in hESCs. Genome-wide, it binds a gene cohort involved in primitive streak formation at gastrulation, including NODAL, BMP4 and WNT3 Comparing TCF7L1-bound sites with those bound by the WNT signaling effector ß-catenin indicates that TCF7L1 acts largely on the WNT signaling pathway. TCF7L1 overlaps less with the pluripotency regulators OCT4 and NANOG than in mouse ESCs. Gain- and loss-of-function studies indicate that TCF7L1 suppresses gene cohorts expressed in the primitive streak. Interestingly, we find that BMP4, another driver of hESC differentiation, downregulates TCF7L1, providing a mechanism of BMP and WNT pathway intersection. Together, our studies indicate that TCF7L1 plays a major role in maintaining hESC pluripotency, which has implications for human development during gastrulation.

Convergence of cMyc and ß-catenin on Tcf7l1 enables endoderm specification.

The molecular machinery that directs formation of definitive endoderm from pluripotent stem cells is not well understood. Wnt/ß-catenin and Nodal signalling have been implicated, but the requirements for lineage specification remain incompletely defined. Here, we demonstrate a potent effect of inhibiting glycogen synthase kinase 3 (GSK3) on definitive endoderm production. We find that downstream of GSK3 inhibition, elevated cMyc and ß-catenin act in parallel to reduce transcription and DNA binding, respectively, of the transcriptional repressor Tcf7l1. Tcf7l1 represses FoxA2, a pioneer factor for endoderm specification. Deletion of Tcf7l1 is sufficient to allow upregulation of FoxA2 in the presence of Activin. In wild-type cells, cMyc contributes by reducing Tcf7l1 mRNA, while ß-catenin acts on Tcf7l1 protein. GSK3 inhibition is further required for consolidation of endodermal fate via upregulation of Sox17, highlighting sequential roles for Wnt signalling. The identification of a cMyc/ß-catenin-Tcf7l1-FoxA2 axis reveals a de-repression mechanism underlying endoderm induction that may be recapitulated in other developmental and patho-logical contexts.

ß-catenin stimulates Tcf7l1 degradation through recruitment of casein kinase 2 in mouse embryonic stem cells.

Activation of the Wnt/ß-catenin signaling pathway by the inhibition of glycogen synthase kinase-3 (GSK-3) will induce Tcf7l1 protein degradation to effectively promote embryonic stem cell (ESC) self-renewal. However, the exact mechanism remains unclear. Here, we found that inhibition of casein kinase 2 (Csnk2) by TBB or DMAT was sufficient to block the reduction of the Tcf7l1 protein induced by CHIR99021, a specific inhibitor of GSK-3. Similarly, downregulation of Csnk2 increased the Tcf7l1 level. In contrast, overexpression of Csnk2 significantly decreased Tcf7l1 protein stability in mouse ESCs. Notably, Csnk2α1 controls Tcf7l1 turnover to a greater degree than the other two isoforms of Csnk2, Csnk2α2 and Csnk2ß, as Csnk2α1-overexpressing mouse ESCs exhibited the lowest level of Tcf7l1. Csnk2α1 interacted with and phosphorylated Tcf7l1. In addition, the association of Csnk2α1 and Tcf7l1 was enhanced by CHIR99021. Our study demonstrated, for the first time, that Csnk2 is involved in Tcf7l1 turnover mediated by the Wnt/ß-catenin signaling pathway. These results expand our understanding of the function and circuit of Wnt/ß-catenin signaling pathway in ESCs.

Wnt suppressor and stem cell regulator TCF7L1 is a sensitive immunohistochemical marker to differentiate testicular seminoma from non-seminomatous germ cell tumor.

The accurate classification and proper identification of testicular germ cell tumors is imperative for treatment selection and clinical prognosis. Although such distinction can often be achieved by microscopic morphology alone, ancillary tests may at times be needed. T-cell factor 7 L1 (TCF7L1, also known as TCF3), a component of the Wnt signaling pathway, plays important roles in embryonic stem cell self-renewal and lineage specification. Here we examined the immunohistochemical expression and diagnostic utility of TCF7L1 in testicular germ cell tumors. Fifty cases of testicular germ cell tumors were collected, including 23 seminomas, 6 embryonal carcinomas, 1 teratoma, 1 choriocarcinoma, and 19 mixed germ cell tumors. The components of the mixed germ cell tumors were seminoma (n = 3), embryonal carcinoma (n = 18), yolk sac tumor (n = 9), teratoma (n = 15), and choriocarcinoma (n = 4). On immunohistochemistry of TCF7L1, only nuclear staining was considered positive. Staining was graded as negative (<5% of tumor cells stained), minimal (5-25% positive), focal (26-50%), and diffuse (>50%). All non-seminomatous components (n = 54) exhibited distinct nuclear expression of TCF7L1 (54/54; 100%). In contrast, no TCF7L1 expression was detected in the majority of seminomatous tumor component (24/26; 92%). Two seminomas (2/26; 8%) exhibited minimal weak nuclear staining (5% and 10%, respectively) for TCF7L1. In conclusion, TCF7L1, highly expressed in non-seminomatous testicular germ cell tumors, might be used as a marker for diagnosis of testicular germ cell tumors, two therapeutically different entities, for better patient management.

TCF7L1 indicates prognosis and promotes proliferation through activation of Keap1/NRF2 in gastric cancer.

Gastric cancer is one of the most common cancers worldwide and is the third leading cause of cancer-related deaths globally. Although significant progress has been made in the diagnosis and treatment for the cancer, less improvement has been made in overall survival rate. Thus, there is an urgent need for a better understanding of the biological aspects of the cancer. The transcription factor transcription factor 7-like 1 (TCF7L1) is an embryonic stem cell signature gene that is upregulated in multiple aggressive cancer types, but its role in gastric cancer has seldom been discussed. In the present study, by using the Cancer Genome Atlas dataset analysis, we demonstrated that patients with higher expression of TCF7L1 could be used to reflect prognosis. An examination of the mechanisms demonstrated that TCF7L1 could positively regulate antioxidant response in gastric cancer cells by positively regulating Keap1/NRF2 [Kelch-like ECH-associated protein 1/nuclear factor (erythroid-derived 2)-like 2] pathway. Collectively, our data demonstrated that TCF7L1 is a novel marker for predicting overall survival of gastric cancer and provided the possible underlying molecular mechanism.

Transcription factor 7-like 1 is involved in hypothalamo-pituitary axis development in mice and humans.

Aberrant embryonic development of the hypothalamus and/or pituitary gland in humans results in congenital hypopituitarism (CH). Transcription factor 7-like 1 (TCF7L1), an important regulator of the WNT/ß-catenin signaling pathway, is expressed in the developing forebrain and pituitary gland, but its role during hypothalamo-pituitary (HP) axis formation or involvement in human CH remains elusive. Using a conditional genetic approach in the mouse, we first demonstrate that TCF7L1 is required in the prospective hypothalamus to maintain normal expression of the hypothalamic signals involved in the induction and subsequent expansion of Rathke's pouch progenitors. Next, we reveal that the function of TCF7L1 during HP axis development depends exclusively on the repressing activity of TCF7L1 and does not require its interaction with ß-catenin. Finally, we report the identification of two independent missense variants in human TCF7L1, p.R92P and p.R400Q, in a cohort of patients with forebrain and/or pituitary defects. We demonstrate that these variants exhibit reduced repressing activity in vitro and in vivo relative to wild-type TCF7L1. Together, our data provide support for a conserved molecular function of TCF7L1 as a transcriptional repressor during HP axis development in mammals and identify variants in this transcription factor that are likely to contribute to the etiology of CH.

TCF7L1 recruits CtBP and HDAC1 to repress DICKKOPF4 gene expression in human colorectal cancer cells.

The T-cell factor/Lymphoid enhancer factor (TCF/LEF; hereafter TCF) family of transcription factors are critical regulators of colorectal cancer (CRC) cell growth. Of the four TCF family members, TCF7L1 functions predominantly as a repressor of gene expression. Few studies have addressed the role of TCF7L1 in CRC and only a handful of target genes regulated by this repressor are known. By silencing TCF7L1 expression in HCT116 cells, we show that it promotes cell proliferation and tumorigenesis in vivo by driving cell cycle progression. Microarray analysis of transcripts differentially expressed in control and TCF7L1-silenced CRC cells identified genes that control cell cycle kinetics and cancer pathways. Among these, expression of the Wnt antagonist DICKKOPF4 (DKK4) was upregulated when TCF7L1 levels were reduced. We found that TCF7L1 recruits the C-terminal binding protein (CtBP) and histone deacetylase 1 (HDAC1) to the DKK4 promoter to repress DKK4 gene expression. In the absence of TCF7L1, TCF7L2 and ß-catenin occupancy at the DKK4 promoter is stimulated and DKK4 expression is increased. These findings uncover a critical role for TCF7L1 in repressing DKK4 gene expression to promote the oncogenic potential of CRCs.

JmjC Domain-containing Protein 6 (Jmjd6) Derepresses the Transcriptional Repressor Transcription Factor 7-like 1 (Tcf7l1) and Is Required for Body Axis Patterning during Xenopus Embryogenesis.

Tcf7l1 (also known as Tcf3) is a bimodal transcription factor that plays essential roles in embryogenesis and embryonic and adult stem cells. On one hand, Tcf7l1 works as transcriptional repressor via the recruitment of Groucho-related transcriptional corepressors to repress the transcription of Wnt target genes, and, on the other hand, it activates Wnt target genes when Wnt-activated ß-catenin interacts with it. However, how its activity is modulated is not well understood. Here we demonstrate that a JmjC-domain containing protein, Jmjd6, interacts with Tcf7l and derepresses Tcf7l. We show that Jmjd6 binds to a region of Tcf7l1 that is also responsible for Groucho interaction, therefore making it possible that Jmjd6 binding displaces the Groucho transcriptional corepressor from Tcf7l1. Moreover, we show that Jmjd6 antagonizes the repression effect of Tcf7l1 on target gene transcription and is able to enhance ß-catenin-induced gene activation and that, vice versa, inhibition of Jmjd6 activity compromises gene activation in both cells and Xenopus early embryos. We also show that jmjd6 is both maternally and zygotically transcribed during Xenopus embryogenesis. Loss of Jmjd6 function causes defects in anterioposterior body axis formation and down-regulation of genes that are involved in anterioposterior axis patterning. The results elucidate a novel mechanism underlying the regulation of Tcf7l1 activity and the regulation of embryonic body axis formation.

Tcf7l1 proteins cell autonomously restrict cardiomyocyte and promote endothelial specification in zebrafish.

Tcf7l1 (formerly Tcf3) proteins are conserved transcription factors whose function as transcriptional repressors is relieved through interactions with ß-catenin. Although the functions of Tcf7l1 proteins have been studied in many developmental contexts, whether this conserved mediator of Wnt signaling is required for appropriate cardiomyocyte (CM) development has not been investigated. We find that Tcf7l1 proteins are necessary during two developmental periods to limit CM number in zebrafish embryos: prior to gastrulation and after the initial wave of CM differentiation. In contrast to partially redundant roles in anterior neural patterning, we find that Tcf7l1a and Tcf7l1b have non-redundant functions with respect to restricting CM specification during anterior mesodermal patterning, suggesting that between the two zebrafish Tcf7l1 paralogs there is a limit to the transcriptional repression provided during early CM specification. Using cell transplantation experiments, we determine that the Tcf7l1 paralogs are required cell autonomously to restrict CM specification and promote endothelial cell (EC) specification, which is overtly similar to the ability of Wnt signaling to direct a transformation between these progenitors in embryonic stem cells. Therefore, these results argue that during anterior-posterior patterning of the mesoderm Tcf7l1 proteins are cell autonomously required to limit Wnt signaling, which balances CM and EC progenitor specification within the anterior lateral plate mesoderm. This study expands our understanding of the in vivo developmental requirements of Tcf7l1 proteins and the mechanisms directing CM development in vertebrates.

MicroRNA-329-3p inhibits the Wnt/ß-catenin pathway and proliferation of osteosarcoma cells by targeting transcription factor 7-like 1.

An important factor in the emergence and progression of osteosarcoma (OS) is the dysregulated expression of microRNAs (miRNAs). Transcription factor 7-like 1 (TCF7L1), a member of the T cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor family, interacts with the Wnt signaling pathway regulator ß-catenin and acts as a DNA-specific binding protein. This study sought to elucidate the impact of the interaction between miR-329-3p and TCF7L1 on the growth and apoptosis of OS and analyze the regulatory expression relationship between miRNA and mRNA in osteosarcoma cells using a variety of approaches. MiR329-3p was significantly downregulated, while TCF7L1 was considerably up-regulated in all examined OS cell lines. Additionally, a clinical comparison study was performed using the TCGA database. Subsequently, the regulatory relationship between miR-329-3p and TCF7L1 on the proliferation and apoptosis of OS cells was verified through in vitro and in vivo experiments. When miR-329-3p was transfected into the OS cell line, the expression of TCF7L1 decreased, the proliferation of OS cells was inhibited, the cytoskeleton disintegrated, and the nucleus condensed to form apoptotic bodies. The expression of proteins that indicate apoptosis increased simultaneously. The cell cycle was arrested in the G0/G1 phase, and the G1/S transition was blocked. The introduction of miR-329-3p also inhibited downstream Cyclin D1 of the Wnt pathway. Xenograft experiments indicated that the overexpression of miR-329-3p significantly inhibited the growth of OS xenografts in nude mice, and the expression of TCF7L1 and c-Myc in tumor tissues decreased. MiR-329-3p was significantly reduced in OS cells and played a suppressive role in tumorigenesis and proliferation by targeting TCF7L1 both in vitro and in vivo. Osteosarcoma cell cycle arrest and pathway inhibition were observed upon the regulation of TCF7L1 by miR-329-3p. Summarizing these results, it can be inferred that miR-329-3p exerts anticancer effects in osteosarcoma by inhibiting TCF7L1.

TCF7L1 Controls the Differentiation of Tuft Cells in Mouse Small Intestine.

Continuous and rapid renewal of the intestinal epithelium depends on intestinal stem cells (ISCs). A large repertoire of transcription factors mediates the correct maintenance and differentiation of ISCs along either absorptive or secretory lineages. In the present study, we addressed the role of TCF7L1, a negative regulator of WNT signalling, in embryonic and adult intestinal epithelium using conditional mouse mutants. We found that TCF7L1 prevents precocious differentiation of the embryonic intestinal epithelial progenitors towards enterocytes and ISCs. We show that Tcf7l1 deficiency leads to upregulation of the Notch effector Rbp-J, resulting in a subsequent loss of embryonic secretory progenitors. In the adult small intestine, TCF7L1 is required for the differentiation of secretory epithelial progenitors along the tuft cell lineage. Furthermore, we show that Tcf7l1 promotes the differentiation of enteroendocrine D- and L-cells in the anterior small intestine. We conclude that TCF7L1-mediated repression of both Notch and WNT pathways is essential for the correct differentiation of intestinal secretory progenitors.

TCF7L1 Regulates LGR5 Expression in Colorectal Cancer Cells.

Mutations in components of the Wnt/ß-catenin signaling pathway drive colorectal cancer (CRC), in part, by deregulating expression of genes controlled by the T-cell factor (TCF) family of transcription factors. TCFs contain a conserved DNA binding domain that mediates association with TCF binding elements (TBEs) within Wnt-responsive DNA elements (WREs). Intestinal stem cell marker, leucine-rich-repeat containing G-protein-coupled receptor 5 (LGR5), is a Wnt target gene that has been implicated in CRC stem cell plasticity. However, the WREs at the LGR5 gene locus and how TCF factors directly regulate LGR5 gene expression in CRC have not been fully defined. Here, we report that TCF family member, TCF7L1, plays a significant role in regulating LGR5 expression in CRC cells. We demonstrate that TCF7L1 binds to a novel promoter-proximal WRE through association with a consensus TBE at the LGR5 locus to repress LGR5 expression. Using CRISPR activation and interference (CRISPRa/i) technologies to direct epigenetic modulation, we demonstrate that this WRE is a critical regulator of LGR5 expression and spheroid formation capacity of CRC cells. Furthermore, we found that restoring LGR5 expression rescues the TCF7L1-mediated reduction in spheroid formation efficiency. These results demonstrate a role for TCF7L1 in repressing LGR5 gene expression to govern the spheroid formation potential of CRC cells.

TCF7L1 Accelerates Smooth Muscle Cell Phenotypic Switching and Aggravates Abdominal Aortic Aneurysms.

Phenotypic switching of vascular smooth muscle cells is a central process in abdominal aortic aneurysm (AAA) pathology. We found that knockdown TCF7L1 (transcription factor 7-like 1), a member of the TCF/LEF (T cell factor/lymphoid enhancer factor) family of transcription factors, inhibits vascular smooth muscle cell differentiation. This study hints at potential interventions to maintain a normal, differentiated smooth muscle cell state, thereby eliminating the pathogenesis of AAA. In addition, our study provides insights into the potential use of TCF7L1 as a biomarker for AAA.

Systematic identification of cancer-associated-fibroblast-derived genes in patients with colorectal cancer based on single-cell sequencing and transcriptomics.

Colorectal cancer (CRC) has a high incidence rate and poor prognosis, and the available treatment approaches have limited therapeutic benefits. Therefore, understanding the underlying mechanisms of occurrence and development is particularly crucial. Increasing attention has been paid to the pathophysiological role of cancer-associated fibroblasts (CAFs) in the heterogeneous tumour microenvironment. CAFs play a crucial role in tumorigenesis, tumour progression and treatment response. However, routine tissue sequencing cannot adequately reflect the heterogeneity of tumours. In this study, single-cell sequencing was used to examine the fibroblast population in CRC. After cluster analysis, the fibroblast population was divided into four subgroups. The distribution and role of these four subgroups in CRC were found to be different. Based on differential gene expression and lasso regression analysis of the main marker genes in these subgroups, four representative genes were obtained, namely, TCF7L1, FLNA, GPX3 and MMP11. Patients with CRC were divided into the low- and high-risk groups using the prognostic risk model established based on the expression of these four genes. The prognosis of patients in different risk groups varied significantly; patients with low-risk scores had a greater response to PDL1 inhibitors, significant clinical benefits and significantly prolonged overall survival. These effects may be attributed to inhibition of the function of T cells in the immune microenvironment and promotion of the function of tumour-associated macrophages.

Serous BMP8A has Clinical Significance in the Ultrasonic Diagnosis of Thyroid Cancer and Promotes Thyroid Cancer Cell Progression.

OBJECTIVE: This study aims to discover a potential cytokine biomarker for early diagnosis of thyroid cancer. METHODS: We employed data mining of The Cancer Genome Atlas (TCGA) and experimentally elucidated its mechanistic contributions. The differential expression genes (DEGs) between thyroid cancer and health population were analyzed with TCGA online bioinformatic tools. The relative expression of Bone Morphogenetic Protein 8A (BMP8A) was determined by real-time PCR in ultrasonic diagnosed thyroid cancer both in vivo and in vitro. The serous BMP8A content was quantified with an ELISA kit. Protein levels of BMP8A, OCLN, ZEB1, EZH2 and ß-Actin were analyzed by Western blot. Cell viability was measured by the MTT assay, and anchorage-independent growth was measured by the soft agar colony formation assay. Cell migrative and invasive capacities were interrogated with transwell chamber assays. RESULTS: We identified aberrantly high expression of BMP8A in thyroid cancer, which was associated with unfavorable prognosis and tumor progression. The serous BMP8A was also significantly up-regulated in thyroid cancer patients. Ectopic over-expression of BMP8A remarkably stimulated cell viability and anchorage-independent growth. Meanwhile, the migrative and invasive capacities were greatly increased in response to BMP8A over-expression. Mechanistically, we characterized the positive correlation between BMP8A and TCF7L1, and forced expression of TCF7L1 induced BMP8A expression in TPC-1 cells. CONCLUSION: In summary, we have identified a novel biomarker for early diagnosis in addition to Ultrasound for thyroid cancer, which is subjected to TCF7L1 regulation.

Compensatory growth renders Tcf7l1a dispensable for eye formation despite its requirement in eye field specification.

The vertebrate eye originates from the eye field, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7l1a is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eye field in tcf7l1a mutants, these fish develop normal eyes revealing a striking ability of the eye to recover from a severe early phenotype. This robustness is not mediated through genetic compensation at neural plate stage; instead, the smaller optic vesicle of tcf7l1a mutants shows delayed neurogenesis and continues to grow until it achieves approximately normal size. Although the developing eye is robust to the lack of Tcf7l1a function, it is sensitised to the effects of additional mutations. In support of this, a forward genetic screen identified mutations in hesx1, cct5 and gdf6a, which give synthetically enhanced eye specification or growth phenotypes when in combination with the tcf7l1a mutation.

Intracellular Ca2+ Homeostasis and Nuclear Export Mediate Exit from Naive Pluripotency.

Progression through states of pluripotency is required for cells in early mammalian embryos to transition away from heightened self-renewal and toward competency for lineage specification. Here, we use a CRISPR mutagenesis screen in mouse embryonic stem cells (ESCs) to identify unexpected roles for nuclear export and intracellular Ca2+ homeostasis during the exit out of the naive state of pluripotency. Mutation of a plasma membrane Ca2+ pump encoded by Atp2b1 increased intracellular Ca2+ such that it overcame effects of intracellular Ca2+ reduction, which is required for naive exit. Persistent self-renewal of ESCs was supported both in Atp2b1-/-Tcf7l1-/- double-knockout ESCs passaged in defined media alone (no LIF or inhibitors) and in wild-type cells passaged in media containing only calcitonin and a GSK3 inhibitor. These new findings suggest a central role for intracellular Ca2+ in safeguarding naive pluripotency.