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[Background]: Fibroblast growth factors (Fgfs) are required for survival and organ formation during embryogenesis. Fgfs often execute their functions redundantly. Previous analysis of Fgf3 mutants revealed effects on inner ear formation and embryonic survival with incomplete penetrance. [Results]: Here, we show that presence of a neomycin resistance gene (neo) replacing the Fgf3 coding region leads to reduced survival during embryogenesis and an increased penetrance of inner ear defects. Fgf3neo/neo mutants showed reduced expression of Fgf4, which is positioned in close proximity to the Fgf3 locus in the mouse genome. Conditional inactivation of Fgf4 during inner ear development on a Fgf3 null background using Fgf3/4 cis mice revealed a redundant requirement between these Fgfs during otic placode induction. In contrast, inactivation of Fgf3 and Fgf4 in the pharyngeal region where both Fgfs are also co-expressed using a Foxg1-Cre driver did not affect development of the pharyngeal arches. However, these mutants showed reduced perinatal survival. [Conclusions]: These results highlight the importance of Fgf signaling during development. In particular, different members of the Fgf family act redundantly to guarantee inner ear formation and embryonic survival., Consejería de Educación, Junta de Castilla y León, Grant/Award Number: CSI143P20; Programa Estratégico Instituto de Biología y Genética Molecular (IBGM), Escalera de Excelencia, Junta de Castilla y León, Grant/Award Numbers: CCVC8485, CLU-2019-02; MEC, Grant/Award Number: BFU2004-00860/BFI

The tyrosine kinase inhibitor lenvatinib is used to treat advanced hepatocellular carcinoma (HCC). Ferroptosis is a type of cell death characterized by the iron-dependent accumulation of lethal lipid reactive oxygen species (ROS). Nuclear factor erythroid-derived 2-like 2 (Nrf2) protects HCC cells against ferroptosis. However, the mechanism of lenvatinib-induced cytotoxicity and the relationships between lenvatinib resistance and Nrf2 are unclear. Thus, we investigated the relationship between lenvatinib and ferroptosis and clarified the involvement of Nrf2 in lenvatinib-induced cytotoxicity. Cell viability, lipid ROS levels, and protein expression were measured using Hep3B and HuH7 cells treated with lenvatinib or erastin. We examined these variables after silencing fibroblast growth factor receptor-4 (FGFR4) or Nrf2 and overexpressing-Nrf2. We immunohistochemically evaluated FGFR4 expression in recurrent lesions after resection and clarified the relationship between FGFR4 expression and lenvatinib efficacy. Lenvatinib suppressed system X

The mechanisms used by embryos to pattern tissues across their axes has fascinated developmental biologists since the founding of embryology. Here, using single-cell technology, we interrogate complex patterning defects and define a Hedgehog (Hh)–fibroblast growth factor (FGF) signaling axis required for anterior mesoderm lineage development during gastrulation. Single-cell transcriptome analysis of Hh-deficient mesoderm revealed selective deficits in anterior mesoderm populations, culminating in defects to anterior embryonic structures, including the pharyngeal arches, heart, and anterior somites. Transcriptional profiling of Hh-deficient mesoderm during gastrulation revealed disruptions to both transcriptional patterning of the mesoderm and FGF signaling for mesoderm migration. Mesoderm-specific Fgf4/Fgf8 double-mutants recapitulated anterior mesoderm defects and Hh-dependent GLI transcription factors modulated enhancers at FGF gene loci. Cellular migration defects during gastrulation induced by Hh pathway antagonism were mitigated by the addition of FGF4 protein. These findings implicate a multicomponent signaling hierarchy activated by Hh ligands from the embryonic node and executed by FGF signals in nascent mesoderm to control anterior mesoderm patterning.

Maturation and synchronisation of heart cells, including cardiomyocytes and fibroblasts, are essential to develop functional biomimetic cardiac tissues for regenerative medicine and drug discovery. Synchronisation of cells in the biomimetic cardiac tissue requires the structural integrity and functional maturation of cardiomyocytes with other cell types. However, it is challenging to synchronise the beating of macroscale cardiac tissues and induce maturation of cardiomyocytes derived from stem cells. Here, we developed a simple assembly technology to modulate cell-cell interactions by combining layer-by-layer (LBL) deposition and centrifugation of cells with collagen type I to control cell-cell interactions for the preparation of cardiac macro tissues (CMTs). We found that maturation of cardiomyocytes in CMTs was largely enhanced by growth factors FGF-4 and ascorbic acid, but synchronisation of cardiac beating required LBL deposition of cardiomyocytes and cardiac fibroblasts in addition to the growth factors during the maturation process. Our findings have important implications because incorporation of cardiac fibroblasts into the cardiomyocyte layer is a prerequisite for synchronised beating of macroscale cardiac tissues in addition to growth factors to facilitate maturation of stem cell-derived cardiomyocytes.

NAFLD represents an increasing health problem in association with obesity and diabetes with no effective pharmacotherapies. Growing evidence suggests that several FGFs play important roles in diverse aspects of liver pathophysiology. Here, we report a previously unappreciated role of FGF4 in the liver.Expression of hepatic FGF4 is inversely associated with NAFLD pathological grades in both human patients and mouse models. Loss of hepatic Fgf4 aggravates hepatic steatosis and liver damage resulted from an obesogenic high-fat diet. By contrast, pharmacological administration of recombinant FGF4 mitigates hepatic steatosis, inflammation, liver damage, and fibrogenic markers in mouse livers induced to develop NAFLD and NASH under dietary challenges. Such beneficial effects of FGF4 are mediated predominantly by activating hepatic FGF receptor (FGFR) 4, which activates a downstream CaOur study identifies FGF4 as a stress-responsive regulator of liver pathophysiology that acts through an FGFR4-AMPK-Caspase 6 signal pathway, shedding light on strategies for treating NAFLD and associated liver pathologies.

Hemodynamic forces have an important role in venous intimal hyperplasia, which is the main cause of arteriovenous fistula dysfunction. Endothelial cells (ECs) constantly exposed to the shear stress of blood flow, converted the mechanical stimuli into intracellular signals, and interacted with the underlying vascular smooth muscle cells (VSMCs). Caveolin-1 is one of the important mechanoreceptors on cytomembrane, which is related to vascular abnormalities. Extracellular signal-regulated kinase1/2 (ERK1/2) pathway is involved in the process of VSMCs proliferation and migration. In the present study, we explore the effects of Caveolin-1-ERK1/2 pathway and uremia toxins on the endothelial cells and VSMCs following shear stress application. Different shear stress was simulated with a ECs/VSMCs cocultured parallel-plate flow chamber system. Low shear stress and oscillating shear stress up-regulated the expression of fibroblast growth factor-4, platelet-derived growth factor-BB, vascular endothelial growth factor-A, ERK1/2 phosphorylation in endothelial cells, and proliferation and migration of VSMCs but down-regulated the Caveolin-1 expression in endothelial cells. Uremia toxin induces the proliferation and migration of VSMCs but not in a Caveolin-1-dependent manner in the static environment. Low shear stress-induced proliferation and migration of VSMCs is inhibited by Caveolin-1 overexpression and ERK1/2 suppression. Shear stress-regulated VSMC proliferation and migration is an endothelial cells-dependent process. Low shear stress and oscillating shear stress exert atherosclerotic influences on endothelial cells and VSMCs. Low shear stress modulated proliferation and migration of VSMCs through Caveolin-1-ERK1/2 pathway, which suggested that Caveolin-1 and ERK1/2 can be used as a new therapeutic target for the treatment of arteriovenous fistula dysfunction.Impact statementVenous intimal hyperplasia is the leading cause of arteriovenous fistula (AVF) dysfunction. This article reports that shear stress-regulated vascular smooth muscle cells (VSMCs) proliferation and migration is an endothelial cell (EC)-dependent process. Low shear stress (LSS) and oscillating shear stress (OSS) exert atherosclerotic influences on the ECs and VSMCs. LSS-induced proliferation and migration of VSMCs is inhibited by Caveolin-1 overexpression and extracellular signal-regulated kinase1/2 (ERK1/2) suppression, which suggested that Caveolin-1 and ERK1/2 can be used as a new therapeutic target for the treatment of AVF dysfunction.

Several members of the FGF family have been identified as potential regulators of glucose homeostasis. We previously reported that a low threshold of FGF-induced FGF receptor 1c (FGFR1c) dimerization and activity is sufficient to evoke a glucose lowering activity. We therefore reasoned that ligand identity may not matter, and that besides paracrine FGF1 and endocrine FGF21, other cognate paracrine FGFs of FGFR1c might possess such activity. Indeed, via a side-by-side testing of multiple cognate FGFs of FGFR1c in diabetic mice we identified the paracrine FGF4 as a potent anti-hyperglycemic FGF. Importantly, we found that like FGF1, the paracrine FGF4 is also more efficacious than endocrine FGF21 in lowering blood glucose. We show that paracrine FGF4 and FGF1 exert their superior glycemic control by targeting skeletal muscle, which expresses copious FGFR1c but lacks β-klotho (KLB), an obligatory FGF21 co-receptor. Mechanistically, both FGF4 and FGF1 upregulate GLUT4 cell surface abundance in skeletal muscle in an AMPKα-dependent but insulin-independent manner. Chronic treatment with rFGF4 improves insulin resistance and suppresses adipose macrophage infiltration and inflammation. Notably, unlike FGF1 (a pan-FGFR ligand), FGF4, which has more restricted FGFR1c binding specificity, has no apparent effect on food intake. The potent anti-hyperglycemic and anti-inflammatory properties of FGF4 testify to its promising potential for use in the treatment of T2D and related metabolic disorders., Fibroblast growth factors are involved in systemic glucose homeostasis and of interest for developing therapies for type 2 diabetes and associated metabolic diseases. Here the authors identify paracrine FGF4 as an anti-hyperglycemic FGF, which targets skeletal muscle to upregulate the glucose transporter GLUT4 cell surface abundance.

Non-alcoholic fatty liver disease (NAFLD) has emerged as a major liver disease increasingly in association with non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). However, there are currently no approved therapies for treating NAFLD and NASH. Fibroblast growth factor 4 (FGF4) has recently been shown as a promising drug candidate for several metabolic diseases.Mice fed a high-fat diet with high fructose/glucose drinking water (HF/HFG, Western-like diet) for 21 weeks were intraperitoneally injected with non-mitogenic recombinant FGF4Administration of rFGF4rFGF4

Mature cardiomyocytes (CMs) obtained from human pluripotent stem cells (hPSCs) have been required for more accurate in vitro modeling of adult-onset cardiac disease and drug discovery. Here, we found that FGF4 and ascorbic acid (AA) induce differentiation of BG01 human embryonic stem cell–cardiogenic mesoderm cells (hESC-CMCs) into mature and ventricular CMs. Co-treatment of BG01 hESC-CMCs with FGF4+AA synergistically induced differentiation into mature and ventricular CMs. FGF4+AA-treated BG01 hESC-CMs robustly released acute myocardial infarction (AMI) biomarkers (cTnI, CK-MB, and myoglobin) into culture medium in response to hypoxic injury. Hypoxia-responsive genes and potential cardiac biomarkers proved in the diagnosis and prognosis of coronary artery diseases were induced in FGF4+AA-treated BG01 hESC-CMs in response to hypoxia based on transcriptome analyses. This study demonstrates that it is feasible to model hypoxic stress in vitro using hESC-CMs matured by soluble factors.

SOX2 is an oncogenic transcription factor overexpressed in nearly half of the basal-like triple-negative breast cancers associated with very poor outcomes. Targeting and inhibiting SOX2 is clinically relevant as high SOX2 mRNA levels are positively correlated with decreased overall survival and progression-free survival in patients affected with breast cancer. Given its key role as a master regulator of cell proliferation, SOX2 represents an important scaffold for the engineering of dominant-negative synthetic DNA-binding domains (DBDs) that act by blocking or interfering with the oncogenic activity of the endogenous transcription factor in cancer cells. We have synthesized an interference peptide (iPep) encompassing a truncated 24 amino acid long C-terminus of SOX2 containing a potential SOX-specific nuclear localization sequence, and the determinants of the binding of SOX2 to the DNA and to its transcription factor binding partners. We found that the resulting peptide (SOX2-iPep) possessed intrinsic cell penetration and promising nuclear localization into breast cancer cells, and decreased cellular proliferation of SOX2 overexpressing cell lines. The novel SOX2-iPep was found to exhibit a random coil conformation predominantly in solution. Molecular dynamics simulations were used to characterize the interactions of both the SOX2 transcription factor and the SOX2-iPep with FGF4-enhancer DNA in the presence of the POU domain of the partner transcription factor OCT4. Predictions of the free energy of binding revealed that the iPep largely retained the binding affinity for DNA of parental SOX2. This work will enable the future engineering of novel dominant interference peptides to transport different therapeutic cargo molecules such as anti-cancer drugs into cells.

Epigenetic aberrations are widespread in cancer, yet the underlying mechanisms and causality remain poorly understood1–3. A subset of gastrointestinal stromal tumours (GISTs) lack canonical kinase mutations but instead have succinate dehydrogenase (SDH) deficiency and global DNA hyper-methylation4,5. Here, we associate this hyper-methylation with changes in genome topology that activate oncogenic programs. To investigate epigenetic alterations systematically, we mapped DNA methylation, CTCF insulators, enhancers, and chromosome topology in KIT-mutant, PDGFRA-mutant and SDH-deficient GISTs. Although these respective subtypes shared similar enhancer landscapes, we identified hundreds of putative insulators where DNA methylation replaced CTCF binding in SDH-deficient GISTs. We focused on a disrupted insulator that normally partitions a core GIST super-enhancer from the FGF4 oncogene. Recurrent loss of this insulator alters locus topology in SDH-deficient GISTs, allowing aberrant physical interaction between enhancer and oncogene. CRISPR-mediated excision of the corresponding CTCF motifs in an SDH-intact GIST model disrupted the boundary between enhancer and oncogene, and strongly upregulated FGF4 expression. We also identified a second recurrent insulator loss event near the KIT oncogene, which is also highly expressed across SDH-deficient GISTs. Finally, we established a patient-derived xenograft (PDX) from an SDH-deficient GIST that faithfully maintains the epigenetics of the parental tumour, including hypermethylation and insulator defects. This PDX model is highly sensitive to FGF receptor (FGFR) inhibition, and more so to combined FGFR and KIT inhibition, validating the functional significance of the underlying epigenetic lesions. Our study reveals how epigenetic alterations can drive oncogenic programs in the absence of canonical kinase mutations, with implications for mechanistic targeting of aberrant pathways in cancers. Gastrointestinal stromal tumours can be initiated by gain-of-function mutations of the KIT or PDGFRA oncogenes but also by loss of the metabolic complex succinate dehydrogenase (SDH), which leads to DNA hypermethylation; this study shows that in SDH-deficient tumours, displacement of CTCF insulators by DNA methylation activates oncogene expression, illustrating how epigenetic alterations can drive oncogenic signalling in the absence of kinase mutations.

Stem cells are unspecialized cells and excellent model in developmental biology and a promising approach to the treatment of disease and injury. In the last 30 years, pluripotent embryonic stem (ES) cells were established from murine and primate sources, and display indefinite replicative potential and the ability to differentiate to all three embryonic germ layers. Despite large efforts in many aspects of rodent and non-rodent pluripotent stem cell culture, a number of diverse challenges remain. Natural and synthetic small molecules (SMs) strategy has the potential to overcome these hurdles. Small molecules are typically fast and reversible that target specific signaling pathways, epigenetic processes and other cellular processes. Inhibition of the transforming growth factor-β (TGF-β/Smad) and fibroblast growth factor 4 (FGF4)/ERK signaling pathways by SB431542 and PD0325901 small molecules, respectively, known as R2i, enhances the efficiency of mouse, rat, and chicken pluripotent stem cells passaging from different genetic backgrounds. Therefore, the application of SM inhibitors of TGF-β and ERK1/2 with leukemia inhibitory factor (LIF) allows the cultivation of pluripotent stem cells in a chemically defined condition. In this review, we discuss recently emerging evidence that dual inhibition of TGF-β and FGF signaling pathways plays an important role in regulating pluripotency in both rodent and non-rodent pluripotent stem cells.

Gastrointestinal stromal tumors (GIST) lacking mutations in KIT/PDGFRA or RAS pathways and retaining an intact SDH complex are usually referred to as KIT/PDGFRA/SDH/RAS‐P WT GIST or more simply quadruple WT GIST (~5% of all GIST). Despite efforts made, no recurrent genetic event in quadruple WT GIST has been identified so far. To further investigate this disease, we performed high throughput copy number analysis on quadruple WT GIST specimens identifying a recurrent focal gain in band 11q13.3 (involving FGF3/FGF4) in 6/8 cases. This event was not found in the other molecular GIST subgroups. FGF3/FGF4 duplication was associated with high expression of FGF4, both at mRNA and protein level, a growth factor normally not expressed in adult tissues or in KIT/PDGFRA‐mutated GIST. FGFR1 was found to be the predominant FGF receptor expressed and phosphorylation of AKT was detected, suggesting that a FGF4‐FGFR1 autocrine loop could stimulate downstream signaling in quadruple WT GIST. Together with the recent reports of quadruple WT cases carrying FGFR1 activating alterations, these findings strengthen the hypothesis of a potential involvement of FGFR pathway deregulation in quadruple WT GIST, which may represent a rationale for novel therapeutic approaches.

In mice, trophoblast stem (TS) cells are derived from the polar trophectoderm of blastocysts. TS cells cultured in the presence of fibroblast growth factor 4 (Fgf4) are in an undifferentiated state and express undifferentiated marker genes such as Cdx2. After removing Fgf4 from the culture medium, TS cells drastically reduce the expression of undifferentiated marker genes, stop cell proliferation, and differentiate into all trophoblast cell subtypes. To clarify the roles of the parental genomes in placentation, we previously established TS cells from androgenetic embryos (AGTS cells). AGTS cells are in the undifferentiated state when cultured with Fgf4 and express undifferentiated marker genes. After removing Fgf4, AGTS cells differentiate into trophoblast giant cells (TGCs), but not into spongiotrophoblast cells, and some of the AGTS cells continue to proliferate. In this study, we investigated the differentiation potency of AGTS cells by analyzing the expression of undifferentiated marker genes and all trophoblast cell subtype-specific genes. After removing Fgf4, some undifferentiated marker genes (Cdx2, Eomes and Elf5) continued to be expressed. Interestingly, TGCs differentiated from AGTS cells also expressed Cdx2, but not Prl3d1. Moreover, the expression of Gcm1 and Synb was induced after the differentiation, indicating that AGTS cells preferentially differentiated into labyrinth progenitor cells. Cdx2 knockdown resulted in increased Prl3d1 expression, suggesting that Fgf4-independent Cdx2 expression inhibited the functional TGCs. Moreover, Fgf4-independent Cdx2 expression was activated by Gab1, one of the paternally expressed imprinted genes via the mitogen-activated protein kinase kinase (MEK)-extracellular signal regulated protein kinase (ERK) pathway. These results suggested that the paternal genome activates the MEK-ERK pathway without the Fgf4 signal, accelerates the differentiation into labyrinth progenitor cells and controls the function of TGCs.

Summary Although pluripotent stem cells can generate various types of differentiated cells, it is unclear why lineage-committed stem/progenitor cells derived from pluripotent stem cells are decelerated and why the differentiation-resistant propensity of embryonic stem cell (ESC)/induced pluripotent stem cell (iPSC)-derived cells is predominant compared with the in vivo equivalents derived from embryonic/adult tissues. In this study, we demonstrated that iPSCs reprogrammed and maintained with three chemical inhibitors of the fibroblast growth factor 4-mitogen-activated protein kinase cascade and GSK3β (3i) could be differentiated into all three germ layers more efficiently than the iPSCs reprogrammed without the 3i chemicals, even though they were maintained with 3i chemicals once they were reprogrammed. Although the iPSCs reprogrammed with 3i had increased numbers of Zscan4-positive cells, the Zscan4-positive cells among iPSCs that were reprogrammed without 3i did not have an accelerated differentiation ability. These observations suggest that 3i exposure during the reprogramming period determines the accelerated differentiation/maturation potentials of iPSCs that are stably maintained at the distinct state., Graphical Abstract, Highlights • iPSCs reprogrammed and maintained with 3i have accelerated differentiation potential • iPSC colonies reprogrammed with 3i contain increased number of Zscan4 (+) cells, Mouse iPSCs reprogrammed and maintained with three chemical inhibitors of the FGF4-MAPK cascade and GSK3β (3i; PD184352, CHIR99021, and SU5402) could be differentiated into all three germ layers efficiently and contain increased numbers of Zscan4, a 2-cell stage marker, positive cells.

Signal transduction networks generate characteristic dynamic activities to process extracellular signals and guide cell fate decisions such as to divide or differentiate. The differentiation of pluripotent cells is controlled by FGF/ERK signaling. However, only a few studies have addressed the dynamic activity of the FGF/ERK signaling network in pluripotent cells at high time resolution. Here, we use live cell sensors in wild-type and Fgf4-mutant mouse embryonic stem cells to measure dynamic ERK activity in single cells, for defined ligand concentrations and differentiation states. These sensors reveal pulses of ERK activity. Pulsing patterns are heterogeneous between individual cells. Consecutive pulse sequences occur more frequently than expected from simple stochastic models. Sequences become more prevalent with higher ligand concentration, but are rarer in more differentiated cells. Our results suggest that FGF/ERK signaling operates in the vicinity of a transition point between oscillatory and non-oscillatory dynamics in embryonic stem cells. The resulting heterogeneous dynamic signaling activities add a new dimension to cellular heterogeneity that may be linked to divergent fate decisions in stem cell cultures.

Our understanding of the spatiotemporal regulation of cardiogenesis is hindered by the difficulties in modeling this complex organ currently by in vitro models. Here we develop a method to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies. Consecutive morphological changes proceed in a self-organizing manner in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4), and the resulting in vitro heart organoid possesses atrium- and ventricle-like parts containing cardiac muscle, conducting tissues, smooth muscle and endothelial cells that exhibited myocardial contraction and action potentials. The heart organoids exhibit ultrastructural, histochemical and gene expression characteristics of considerable similarity to those of developmental hearts in vivo. Our results demonstrate that this method not only provides a biomimetic model of the developing heart-like structure with simplified differentiation protocol, but also represents a promising research tool with a broad range of applications, including drug testing., Our understanding of the development of the heart has been limited by a lack of in vitro cellular models. Here, the authors treat mouse embryonic stem cell-derived embryoid bodies with laminin-entactin (to mimic the developing microenvironment) and FGF4 to form heart organoids, with atrial and ventricular-like parts.

Background The present study aimed to explore the functions and molecular mechanisms of miR-511 in breast cancer. Methods A quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect miR-511 levels in breast cancer tissues; a chi-squared test was used to analyze the relationship between miR-511 expression level and pathological parameters of breast cancer patients; the proliferation of breast cancer cell lines MDA-MB-231 and MCF-7 was determined by the cell counting kit-8 (CCK-8) assay; migration was determined by scratch wound healing assay and transwell assay; TargetScan was used to predict the binding site between the 3'-untranslated region (3'-UTR) of fibroblast growth factor 4 (FGF4) and miR-511; and qRT-PCR, western blot and a luciferase reporter gene assay were conducted to further validate the targeting relationship between miR-511 and FGF4. Results The expression level of miR-511 was lower in breast cancer tissues than that in adjacent normal tissues. Low expression of miR-511 was associated with larger tumor size, lymph node metastasis and short survival time. In vitro experiments showed that miR-511 modulated the proliferation and metastasis of breast cancer cells. It was also confirmed that miR-511 directly targeted 3'-UTR of FGF4 and reduced its expression, and FGF4 overexpression reversed the effect of miR-511 on the malignant phenotypes of breast cancer cells. Conclusions The results obtained in the present study demonstrate that miR-511 inhibits breast cancer proliferation and metastasis by down-regulating FGF4 expression, which may be helpful in the development of new treatment strategies for breast cancer.

BACKGROUND The aim of this study was to assess the expression and mechanisms of fibroblast growth factor 4 in polydactyly of the thumb induced by cytarabine. MATERIAL AND METHODS Rats were intraperitoneally injected with cytarabine at different gestation periods (12.5 days, 13.5 days, and 14.5 days) to establish a polydactyly of the thumb model. Then, the expression of FGF4 in polydactyly was studied by whole-mount in situ hybridization. We used hematoxylin & eosin stain and cartilage stain to investigate the development of the skeleton and tissues in the embryo. Pictures were taken to determine the general shape of the deformity, then X-rays were taken to detect bone distortion of the rats born with a congenital malformation. RESULTS In the experimental group (11.5 days, 12.5 days, 13.5 days, and 14.5 days), whole-mount in situ hybridization showed that the FGF4 expression at the tip of the embryonic limb bud was significantly increased compared with the control group and FGF4 was distributed in a wider range and lasted longer than in the control group (P

During embryonic development and tissue homeostasis, reproducible proportions of differentiated cell types are specified from populations of multipotent precursor cells. Molecular mechanisms that enable both robust cell-type proportioning despite variable initial conditions in the precursor cells, and the re-establishment of these proportions upon perturbations in a developing tissue remain to be characterized. Here, we report that the differentiation of robust proportions of epiblast-like and primitive endoderm-like cells in mouse embryonic stem cell cultures emerges at the population level through cell-cell communication via a short-range fibroblast growth factor 4 (FGF4) signal. We characterize the molecular and dynamical properties of the communication mechanism and show how it controls both robust cell-type proportioning from a wide range of experimentally controlled initial conditions, as well as the autonomous re-establishment of these proportions following the isolation of one cell type. The generation and maintenance of reproducible proportions of discrete cell types is a new function for FGF signaling that might operate in a range of developing tissues., Summary: Experiments in embryonic stem cells reveal how the coupling of cell fate decisions in a population leads to the self-organized differentiation of stable ratios of cell types.

The formation of the blastocyst during mammalian development involves the segregation of two populations of cells with unequal potential: pluripotent cells of the inner cell mass (ICM) and multipotent cells of the trophectoderm (TE). ICM cells maintain the capacity to give rise to all cells represented in the organism, while TE cells, which represent the first lineage to emerge during development, are capable of differentiating into trophoblast lineages of the placenta. The ICM and TE are both essential for development. The ICM is genetically programmed to generate all cells of the embryo proper, while the TE forms extraembryonic trophoblast lineages and is required for implantation of the embryo and maternal-fetal exchange of nutrients and waste. Embryonic stem (ES) cells, which can be derived from the ICM of blastocysts in the presence of external signals such as LIF, can self-renewal indefinitely, and because they can differentiate into all cells of the organism, ES cells are a widely used in vitro model to study genetics and development. Trophoblast stem (TS) cells can be derived from the TE of blastocyst stage embryos in the presence of FGF4, and like ES cells, TS cells are also able to self-renew indefinitely. Because TS cells can differentiate into epithelial lineages of the trophoblast, TS cells are an ideal in vitro model to study the biology of the trophoblast. In this chapter, we describe protocols for simultaneous derivation of ES cells and TS cells from mouse blastocysts and culture conditions that promote self-renewal of hybrid ESC/TSC colonies. These protocols are sufficient for efficient derivation of hybrid ESC/TSC colonies.

Pluripotent cells in the inner cell mass (ICM) or epiblast of mammalian embryos exhibit the capacity to differentiate into all cells represented in the three germ layers. Embryonic stem (ES) cells can be derived from the ICM of preimplantation stage blastocysts, while epiblast stem cells (EpiSCs) can be derived from the epiblast of postimplantation embryos or preimplantation stage embryos. The ability to derive distinct types of pluripotent cells from blastocyst-stage embryos suggests that optimization of culture conditions can promote self-renewal of various stem cell populations. Moreover, because mouse EpiSCs resemble human pluripotent stem (hPS) cells, EpiSCs are a useful model to study common and divergent mechanisms of self-renewal between orthologous species. In addition, studies have demonstrated that haploid embryos and ES cells can be derived from chemically activated oocytes. Here, we describe a protocol for deriving maternal (parthenogenetic/gynogenetic) EpiSCs (maEpiSCs) from haploid blastocyst-stage embryos. This protocol is suitable to establish an experimental model for the study of mechanisms of EpiSC self-renewal and differentiation.

Premature degeneration of the intervertebral disc and its association with specific chondrodystrophic dog breeds has been recognized for over a century. Several lines of evidence including disease breed predisposition, studies suggesting heritability of premature intervertebral disc degeneration (IVDD) and association of a dog chromosome 12 (CFA 12) locus with intervertebral disc calcification have strongly supported a genetic component in IVDD in dogs. Recent studies documenting association of IVDD with an overexpressing FGF4 retrogene on CFA 12 have opened up new areas of investigation to further define the pathophysiology of premature IVDD. While preliminary data from studies investigating FGF4 retrogenes in IVDD implicate FGF4 overexpression as a major disease factor, they have also highlighted knowledge gaps in our understanding of intervertebral disc herniation which is a complex and multifactorial disease process.

Gastrointestinal stromal tumours that are wild type for KIT and PDGFRA are referred to as WT GISTs. Of these tumours, SDH-deficient (characterized by the loss of SDHB) and quadruple WT GIST (KIT/PDGFRA/SDH/RAS-P WT) subgroups were reported to display a marked overexpression of FGF4, identifying a putative common therapeutic target for the first time. In SDH-deficient GISTs, methylation of an FGF insulator region was found to be responsible for the induction of FGF4 expression. In quadruple WT, recurrent focal duplication of FGF3/FGF4 was reported; however, how it induced FGF4 expression was not investigated. To assess whether overexpression of FGF4 in quadruple WT could be driven by similar epigenetic mechanisms as in SDH-deficient GISTs, we performed global and locus-specific (on FGF4 and FGF insulator) methylation analyses. However, no epigenetic alterations were detected. Conversely, we demonstrated that in quadruple WT GISTs, FGF4 expression and the structure of the duplication were intimately connected, with the copy of FGF4 closer to the ANO1 super-enhancer being preferentially expressed. In conclusion, we demonstrated that in quadruple WT GISTs, FGF4 overexpression is not due to an epigenetic mechanism but rather to the specific genomic structure of the duplication. Even if FGF4 overexpression is driven by different molecular mechanisms, these findings support an increasing biologic relevance of the FGFR pathway in WT GISTs, both in SDH-deficient and quadruple WT GISTs, suggesting that it may be a common therapeutic target.

Two transcribed retrocopies of the fibroblast growth factor 4 (FGF4) gene have previously been described in the domestic dog. An FGF4 retrocopy on chr18 is associated with disproportionate dwarfism, while an FGF4 retrocopy on chr12 is associated with both disproportionate dwarfism and intervertebral disc disease (IVDD). In this study, whole-genome sequencing data were queried to identify other FGF4 retrocopies that could be contributing to phenotypic diversity in canids. Additionally, dogs with surgically confirmed IVDD were assayed for novel FGF4 retrocopies. Five additional and distinct FGF4 retrocopies were identified in canids including a copy unique to red wolves (Canis rufus). The FGF4 retrocopies identified in domestic dogs were identical to domestic dog FGF4 haplotypes, which are distinct from modern wolf FGF4 haplotypes, indicating that these retrotransposition events likely occurred after domestication. The identification of multiple, full length FGF4 retrocopies with open reading frames in canids indicates that gene retrotransposition events occur much more frequently than previously thought and provide a mechanism for continued genetic and phenotypic diversity in canids.

The human stomach contains two primary domains: the corpus, which contains the fundic epithelium, and the antrum. Each of these domains has distinct cell types and functions, and therefore each presents with unique disease pathologies. Here, we detail two protocols to differentiate human pluripotent stem cells (hPSCs) into human gastric organoids (hGOs) that recapitulate both domains. Both protocols begin with the differentiation of hPSCs into definitive endoderm (DE) using activin A, followed by the generation of free-floating 3D posterior foregut spheroids using FGF4, Wnt pathway agonist CHIR99021 (CHIR), BMP pathway antagonist Noggin, and retinoic acid. Embedding spheroids in Matrigel and continuing 3D growth in epidermal growth factor (EGF)-containing medium for 4 weeks results in antral hGOs (hAGOs). To obtain fundic hGOs (hFGOs), spheroids are additionally treated with CHIR and FGF10. Induced differentiation of acid-secreting parietal cells in hFGOs requires temporal treatment of BMP4 and the MEK inhibitor PD0325901 for 48 h on protocol day 30. In total, it takes ~34 d to generate hGOs from hPSCs. To date, this is the only approach that generates functional human differentiated gastric cells de novo from hPSCs.

FGF signaling is known to play a critical role in the specification of primitive endoderm (PrE) and epiblast (Epi) from the inner cell mass (ICM) during mouse preimplantation development, but how FGFs synergize with other growth factor signaling pathways is unknown. Because PDGFRα signaling has also been implicated in the PrE, we investigated the coordinate functions of PDGFRα together with FGFR1 or FGFR2 in PrE development. PrE development was abrogated in Pdgfra; Fgfr1 compound mutants, or significantly reduced in Pdgfra; Fgfr2 or Pdgfra(PI3K); Fgfr2 compound mutants. We provide evidence that both Fgfr2 and Pdgfra play roles in PrE cell survival while Fgfr1 controls PrE cell specification. Our results suggest a model where FGFR1-engaged ERK1/2 signaling governs PrE specification while PDGFRα-and by analogy possibly FGFR2- engaged PI3K signaling regulates PrE survival and positioning in the embryo. Together, these studies indicate how multiple growth factors and signaling pathways can cooperate in preimplantation development.

Summary: This issue reports two studies, one by Hatlen and colleagues and the other by Kim and colleagues, that detail the drug-development journey of the FGF19–FGFR4 inhibitor fisogatinib (BLU-554), from identification of the drug to preclinical validation studies to finally the results of the proof-of-concept first-in-human phase I trial of this potent and selective, type I irreversible inhibitor of FGFR4. Moreover, Hatlen and colleagues also report a resistance mechanism acquired after therapy that targets selective FGFR4 inhibition, which validates FGF as a specific target in hepatocellular cancer. See related article by Hatlen et al., p. 1686. See related article by Kim et al., p. 1696.