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Summary Mouse embryonic stem cells (ESCs) differentiate into multiple cell types during organismal development. Fibroblast growth factor 4 (FGF4) signaling induces differentiation from ESCs via the phosphorylation of downstream molecules such as mitogen-activated protein kinase/extracellular signal-related kinase (MEK) and extracellular signal-related kinase 1/2 (ERK1/2). The FGF4-MEK-ERK1/2 pathway is inhibited to maintain ESCs in the undifferentiated state. However, the inhibitory mechanism of the FGF4-MEK-ERK1/2 pathway in ESCs is uncharacterized. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification characterized by the attachment of a single N-acetylglucosamine (GlcNAc) to the serine and threonine residues of nuclear or cytoplasmic proteins. Here, we showed that the O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ phosphorylation (activation) and, consequently, the FGF4-PKCζ-MEK-ERK1/2 pathway in ESCs. Our results demonstrate the mechanism for the maintenance of the undifferentiated state of ESCs via the inhibition of the FGF4-PKCζ-MEK-ERK1/2 pathway by O-GlcNAcylation on PKCζ., Graphical Abstract, Highlights • PKCζ activates the MEK-ERK1/2 pathway by FGF4 stimulation • O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ activation in ESCs • FGF4-PKCζ-MEK-ERK1/2 pathway is inhibited by O-GlcNAc on PKCζ in ESCs, In this article, Nishihara and colleagues show that PKCζ is modified by O-GlcNAc and that O-GlcNAc on PKCζ inhibits PKCζ phosphorylation (activation) in mouse embryonic stem cells (ESCs). Phosphorylated PKCζ phosphorylates MEK-ERK1/2 via FGF4 stimulation, and phosphorylated ERK1/2 induces ESC differentiation. Inhibition of PKCζ phosphorylation by O-GlcNAc resulted in the inhibition of the FGF4 signal in ESCs.

Mouse embryonic stem cells (mESCs) can be maintained in a pluripotent state when cultured with 2 inhibitors (2i) of extracellular signal-regulated kinase (MEK) and glycogen synthase kinase-3 (GSK3), and Royan 2 inhibitors (R2i) of FGF4 and TGFβ. The molecular mechanisms that control ESC self-renewal and pluripotency are more important for translating stem cell technologies to clinical applications. In this study, we used the shotgun proteomics technique to compare the proteome of the ground state condition (R2i- and 2i-grown cells) to that of serum. Out of 1749 proteins identified, 171 proteins were differentially expressed (p0.05) in the 2i, R2i, and serum samples. Gene ontology (GO) analysis of differentially abundant proteins showed that the focal adhesion signaling pathway significantly down-regulated under ground state conditions. mESCs had highly adhesive attachment under the serum condition, whereas in the 2i and R2i culture conditions, a loss of adhesion was observed and the cells were rounded and grew in compact colonies on gelatin. Quantitative RT-PCR showed reduced expression of the integrins family in the 2i and R2i conditions. The serum culture had more prominent phosphorylation of focal adhesion kinase (FAK) compared to 2i and R2i cultures. Activity of the extracellular signal-regulated kinase (ERK)1/2 decreased in the 2i and R2i cultures compared to serum. Activation of integrins by Mn

Odontogenesis is guided by a complex signaling cascade in which several molecules, including FGF2-4, ensure all dental groups development and specificity. Most of the data on odontogenesis derives from rodents, which does not have all dental groups. Didelphis albiventris is an opossum with the closest dentition to humans, and the main odontogenesis stages occur when the newborns are in the pouch. In this study, D. albiventris postnatals were used to characterize the main stages of their molars development; and also to establish FGF2, FGF3 and FGF4 expression pattern. D. albiventris postnatals were processed for histological and indirect immunoperoxidase analysis of the tooth germs. Our results revealed similar dental structures between D. albiventris and mice. However, FGF2, FGF3 and FGF4 expression patterns were observed in a larger number of dental structures, suggesting broader functions for these molecules in this opossum species. The knowledge of the signaling that determinates odontogenesis in an animal model with complete dentition may contribute to the development of therapies for the replacement of lost teeth in humans. This study may also contribute to the implementation of D. albiventris as model for Developmental Biology studies.

ETS2 repressor factor (ERF) is a ubiquitous transcriptional repressor regulated by Extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Homozygous deletion of Erf in mice blocks chorionic trophoblast differentiation, resulting in the failure of chorioallantoic fusion and subsequent embryo death. Fibroblast growth factor (FGF) signaling is important for proper trophoblast stem cell (TSC) differentiation and development of the hemochorial placenta. Lack of Fgf2 promotes TSC differentiation, while FGF4 or FGF2 is required for murine TSC maintenance. Here, we show that low in vivo Fgf2 mRNA abundance occurs in patches of placental chorion cells and ex vivo in TSCs. This expression is repressed via direct interaction of ERF with the Fgf2 transcription unit is increased in the absence of ERF, and is decreased in the presence of an ERF mutant resistant to ERK phosphorylation. Thus, FGF2 inhibition by ERF appears to be necessary for proper chorionic TSC differentiation, and may account for the block of chorionic trophoblast differentiation in Erf-knockout animals. The differentiation of ERF-overexpressing TSC lines also suggests that ERF may have an FGF2-independent effect during the commitment towards syncytiotrophoblasts.

Chondrodystrophy results in predictable and progressive biochemical and structural changes to the intervertebral disc, resulting in early onset degeneration and dystrophic mineralization of the disc. Accelerated degeneration and mineralization of the intervertebral disc are common in multiple dog breeds and can result in compromised function, herniation, pain, and a variety of neurological sequelae. A mutation responsible for chondrodystrophy in dogs has been identified as an aberrant fibroblast growth factor 4 ( FGF4) retrogene insertion on chromosome 12 (CFA12) and is associated with short stature of the Nova Scotia Duck Tolling Retriever. Segregation of the CFA12 FGF4 retrogene in this dog breed provides an opportunity to examine the effect of retrogene presence on radiographic and histologic appearance of chondrodystrophic disc degeneration within a single breed. Here we found that in the intervertebral discs isolated from 2 dogs with the CFA12 FGF4 genotype, the nucleus pulposus was largely replaced by cartilaginous tissue, and physaliferous notochordal cells were rarely if ever identified. These findings are in contrast to the normal histologic findings in 2 breed-matched dogs lacking the mutation. The findings are consistent with premature chondroid degeneration of the intervertebral disc and suggest that the presence of the CFA12 FGF4 retrogene is sufficient to cause the chondrodystrophic phenotype.

B-cell activating factor (BAFF) is found to be associated with the histological severity of nonalcoholic steatohepatitis (NASH). BAFF was also found to have a protective role in hepatic steatosis via down regulating the expression of steatogenesis genes and enhancing steatosis in hepatocytes through BAFF-R. However, the roles of BAFF during liver regeneration are not well defined. In this study, C57/B6 mice with 70% partial hepatectomy were used as a liver regeneration model. BAFF expression was determined by enzyme immunoassay, and anti-BAFF-neutralizing antibodies were administered to confirm the effects of BAFF on liver regeneration. Western blotting, immunohistochemistry, and florescence staining determined the expression of B-cell CCL/lymphoma 10 (BCL10). The angiogenesis promoting capability was evaluated after the transfection of cells with siRNA targeting BCL10 expression, and the role of NF-&kappa, B was assessed. The results revealed that the BAFF and BCL10 levels were upregulated after partial hepatectomy. Treatment with anti-BAFF-neutralizing antibodies caused death in mice that were subjected to 70% partial hepatectomy within 72 h. In vitro, recombinant BAFF protein did not enhance hepatocyte proliferation, however, transfection with BCL10 siRNA arrested hepatocytes at the G2/M phase. Interestingly, conditioned medium from BAFF-treated hepatocytes enhanced angiogenesis and endothelial cell proliferation. Moreover, Matrix metalloproteinase-9 (MMP-9), Fibroblast growth factor 4 (FGF4), and Interleukin-8 (IL-8) proteins were upregulated by BAFF through BCL10/NF-&kappa, B signaling. In mice that were treated with anti-BAFF-neutralizing antibodies, the microvessel density (MVD) of the remaining liver tissues and liver regeneration were both reduced. Taken together, our study demonstrated that an increased expression of BAFF and activation of BCL10/NF-&kappa, B signaling were involved in hepatocyte-driven angiogenesis and survival during liver regeneration.

Activation of the ERK signalling pathway is essential for the differentiation of the inner cell mass (ICM) during mouse preimplantation development. We show here that ERK phosphorylation is present in ICM precursor cells, in differentiated Primitive Endoderm (PrE) cells as well as in the mature, formative state Epiblast (Epi). We further show that DUSP4 and ETV5, factors often involved in negative feedback loops of the FGF pathway are differently regulated. While DUSP4 presence clearly depends on ERK phosphorylation in PrE cells, ETV5 localises mainly to Epi cells. Unexpectedly, ETV5 accumulation does not depend on direct activation by ERK but requires NANOG activity. Indeed ETV5, like Fgf4 expression, is not present in Nanog mutant embryos. Our results lead us to propose that in pluripotent early Epi cells, NANOG induces the expression of both Fgf4 and Etv5 to enable the differentiation of neighbouring cells into PrE while protecting the Epi identity from autocrine signalling.

Two FGF4 retrogenes on chromosomes 12 (12-FGF4RG) and 18 (18-FGF4RG) contribute to short-limbed phenotypes in dogs. 12-FGF4RG has also been associated with intervertebral disc disease (IVDD). Both of these retrogenes were found to be widespread among dog breeds with allele frequencies ranging from 0.02 to 1, however, their additive contribution to disease is unknown. Surgical cases of IVDD (n = 569) were evaluated for age of onset, disc calcification, and genotypes for the FGF4 retrogenes. Multivariable linear regression analysis identified the presence of one or two copies of 12-FGF4RG associated with significantly younger age at first surgery in a dominant manner. 18-FGF4RG had only a minor effect in dogs with one copy. Multivariable logistic regression showed that 12-FGF4RG had an additive effect on radiographic disc calcification, while 18-FGF4RG had no effect. Multivariable logistic regression using mixed breed cases and controls identified only 12-FGF4RG as highly associated with disc herniation in a dominant manner (Odds Ratio, OR, 18.42, 95% Confidence Interval (CI) 7.44 to 50.26, P &lt, 0.001). The relative risk for disc surgery associated with 12-FGF4RG varied from 5.5 to 15.1 within segregating breeds and mixed breeds. The FGF4 retrogene on CFA12 acts in a dominant manner to decrease the age of onset and increase the overall risk of disc disease in dogs. Other modifiers of risk may be present within certain breeds, including the FGF4 retrogene on CFA18.

The present study aimed to investigate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) that had been pretreated with pioglitazone and/or rosiglitazone on the growth and proliferation rate of MCF-7 cells. The adhesive interaction between the BMSCs and the MCF-7 cancer cells revealed that the pretreatment of BMSCs with a combination of two types of thiazolidinedione drug reduced the growth and proliferation rate of the MCF-7 cells. The proliferation rate of the MCF-7 cells could also be reduced by the non-adhesive interaction of the cancer cells with BMSCs pretreated with pioglitazone and/or rosiglitazone. The growth and proliferation rate reduction effects on the MCF-7 cells may be attributed to the reduction in the protein level of fibroblast growth factor 4 (FGF4) in the conditioned medium of the pretreated BMSCs. The evidence that the low protein level of FGF4 in the conditioned medium of the pretreated BMSCs perturbed the proliferation rate of the MCF-7 cells by reducing the levels of Ki-67 and proliferating cell nuclear antigen transcripts in the cancer cells was also demonstrated in the present study using a FGF4-neutralizing antibody. All the above findings demonstrate that future studies on the correlation between FGF4 and pretreated BMSCs would be beneficial.

International audience; During development, interactions between transcription factors control the specification of different cell fates. The regulatory networks of genetic interactions often exhibit multiple stable steady states; such multistability provides a common dynamical basis for differentiation. During early murine embryogenesis, cells from the inner cell mass (ICM) can be specified in epiblast (Epi) or primitive endoderm (PrE). Besides the intracellular gene regulatory network, specification is also controlled by intercellular interactions involving Erk signaling through extracellular Fgf4. We previously proposed a model that describes the gene regulatory network and its interaction with Erk signaling in ICM cells. The model displays tristability in a range of Fgf4 concentrations and accounts for the self-organized specification process observed in vivo. Here, we further investigate the origin of tristability in the model and analyze in more detail the specification process by resorting to a simplified two-cell model. We also carry out simulations of a population of 25 cells under various experimental conditions to compare their outcome with that of mutant embryos or of embryos submitted to exogenous treatments that interfere with Fgf signaling. The results are analyzed by means of bifurcation diagrams. Finally, the model predicts that heterogeneities in extracellular Fgf4 concentration play a primary role in the spatial arrangement of the Epi/PrE cells in a salt-and-pepper pattern. If, instead of heterogeneities in extracellular Fgf4 concentration, internal fluctuations in the levels of expression of the transcription factors are considered as a source of randomness, simulations predict the occurrence of unrealistic switches between the Epi and the PrE cell fates, as well as the evolution of some cells toward one of these states without passing through the previous ICM state, in contrast to what is observed in vivo.

Mouse trophoblast stem cells (TSCs) form colonies of different sizes and morphologies, which might reflect their degrees of differentiation. Therefore, each colony type can have a characteristic gene expression profile; however, the expression levels of internal reference genes may also change, causing fluctuations in their estimated gene expression levels. In this study, we validated seven housekeeping genes by using a geometric averaging method and identified Gapdh as the most stable gene across different colony types. Indeed, when Gapdh was used as the reference, expression levels of Elf5, a TSC marker gene, stringently classified TSC colonies into two groups: a high expression groups consisting of type 1 and 2 colonies, and a lower expression group consisting of type 3 and 4 colonies. This clustering was consistent with our putative classification of undifferentiated/differentiated colonies based on their time-dependent colony transitions. By contrast, use of an unstable reference gene (Rn18s) allowed no such clear classification. Cdx2, another TSC marker, did not show any significant colony type-specific expression pattern irrespective of the reference gene. Selection of stable reference genes for quantitative gene expression analysis might be critical, especially when cell lines consisting of heterogeneous cell populations are used.

During the past decade, the application of genomic analysis to liver tumors has provided extensive data concerning tumor phenotypes, signatures, outcomes, and prognosis. In this report the authors describe a middle-aged man without known risk factors for liver disease or hepatocellular carcinoma (HCC) who developed a 19-cm HCC in his right lobe. The underlying liver was normal histologically except for multifocal glycogenotic foci similar to those found in experimental chemical carcinogenesis. Precision genomic analysis of this tumor disclosed five alterations with amplifications of genes CCNE1, FGF3 and FGF4, MYCL1, and ARID1A. The roles of these gene mutations and their potential effects in carcinogenesis in this case are discussed.

Fibroblast growth factor (FGF) 5 regulates the development and periodicity of hair follicles, which can affect hair traits. Loss-of-function mutations associated with long-hair phenotypes have been described in several mammalian species. Sheep is an important economic animal, however, the evolution characterizations and biological mechanism of oFGF5 (Ovis aries FGF5) gene are still poorly understood. In this study, oFGF5 gene was obtained by resequencing the whole genome of three Dorper sheep and RACE of two Kazakh sheep FGF5. We proposed FGF5 was phylogenetically related to FGF4 family and oFGF5 clearly orthologed to goat FGF5. Six loci were found from the positive selection results of FGF5 and half of them located on signal peptide. The basically similar rates of function-altering substitutions in sheep and goat lineage and the rest of the mammalian lineage of 365 SNPs indicated that the FGF5 gene was quite conservative during evolution. Homology modeling of the oFGF5 suggested that it has a highly conserved FGF superfamily domain containing 10 β-strands. Furthermore, the protein-protein docking analysis revealed that oFGF5 have the potential to form heterodimers with oFGFR1, the predicted interaction interface of FGF5-FGFR1 heterodimer was formed mainly by residues from FGF superfamily domain. Our observations suggested the evolutionary and structural biology features of oFGF5 might be relevant to its function about hair follicle development and modulating hair growth, and we confirmed our speculation by using the FGF5 gene editing sheep produced by CRISPR/Cas9 technology.

The neural crest (NC) is a multipotent embryonic cell population that generates distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

Certain bone and soft tissue (BST) tumours harbour a chromosomal translocation [t(6;22)(p21;q12)], which fuses the Ewing's sarcoma (EWS) gene at 22q12 with the octamer-binding transcription factor 4 (Oct-4) gene at 6p21, resulting in the chimeric EWS-Oct-4 protein that possesses high transactivation ability. Although abnormal activation of signalling pathways can lead to human cancer development, the pathways underlying these processes in human BST tumours remain poorly explored. Here, we investigated the functional significance of fibroblast growth factor (FGF) signalling in human BST tumours. To identify the gene(s) involved in the FGF signalling pathway and potentially regulated by EWS-Oct-4 (also called EWS-POU5F1), we performed RNA-Seq analysis, electrophoretic mobility shift assays, chromatin immunoprecipitation assays, and xenograft assays. Treating GBS6 or ZHBTc4 cells-expressing EWS-Oct-4 with the small molecule FGF receptor (FGFR) inhibitors PD173074, NVPBGJ398, ponatinib, and dovitinib suppressed cellular proliferation. Gene expression analysis revealed that, among 22 Fgf and four Fgfr family members, Fgf-4 showed the highest upregulation (by 145-fold) in ZHBTc4 cells-expressing EWS-Oct-4. Computer-assisted analysis identified a putative EWS-Oct-4-binding site at +3017/+3024, suggesting that EWS-Oct-4 regulates Fgf-4 expression in human BST tumours. Fgf-4 enhancer constructs showed that EWS-Oct-4 transactivated the Fgf-4 gene reporter in vitro, and that overexpression of EWS-Oct-4 stimulated endogenous Fgf-4 gene expression in vivo. Finally, PD173074 significantly decreased tumour volume in mice. Taken together, these data suggest that FGF-4 signalling is involved in EWS-Oct-4-mediated tumorigenesis, and that its inhibition impairs tumour growth in vivo significantly.

NF-Y is a bifunctional transcription factor capable of activating or repressing transcription. NF-Y specifically recognizes CCAAT box motifs present in many eukaryotic promoters. The mechanisms involved in regulating its activity are poorly understood. Previous studies have shown that the FGF-4 promoter is regulated positively by its CCAAT box and NF-Y in embryonal carcinoma (EC) cells where the distal enhancer of the FGF-4 gene is active. Here, we demonstrate that the CCAAT box functions as a negative cis-regulatory element when cis-regulatory elements of the FGF-4 enhancer are disrupted, or after EC cells differentiate and the FGF-4 enhancer is inactivated. We also demonstrate that NF-Y mediates the repression of the CCAAT box and that NF-Y associates with the endogenous FGF-4 gene in both EC cells and EC-differentiated cells. Importantly, we also determined that the orientation and the position of the CCAAT box are critical for its role in regulating the FGF-4 promoter. Together, these studies demonstrate that the distal enhancer of the FGF-4 gene determines whether the CCAAT box of the FGF-4 promoter functions as a positive or a negative cis-regulatory element. In addition, these studies are consistent with NF-Y playing an architectural role in its regulation of the FGF-4 promoter.

Cardiovascular diseases along with MI (myocardial infarction) lead to regional ischaemia and hypoxic conditions, which prevail after infarction. Diminished O

Embryonic morphogenesis of a complex organism requires proper regulation of patterning and directional growth. The Planar Cell Polarity (PCP) signaling is emerging as a critical evolutionarily conserved mechanism whereby directional information is conveyed. PCP is thought to be established by global cues, and recent studies have revealed an instructive role of Wnt signaling gradient in epithelia tissues of both invertebrates and vertebrates. However, it remains unclear whether Wnt/PCP signaling is coordinately regulated with embryonic patterning during morphogenesis. Here in the mammalian developing limbs, we find that the AER-derived Fgfs required for limb patterning regulates PCP along the proximal-distal axis in a Wnt5a-dependent manner. We demonstrate with genetic evidence that Wnt5a gradient acts as a global cue that is instructive in establishing PCP in the limb mesenchyme, while Wnt5a also plays a permissive role to allow Fgf signaling to orient PCP. Our results indicate that limb morphogenesis is critically regulated by coordination of directional growth and patterning through integrating Wnt5a and Fgf signaling in PCP regulation.

In the present study, we aimed to determine the prognostic impact and clinicopathological feature of FGF4 amplification in patients with esophageal squamous cell carcinoma (ESCC). Fluorescence in situ hybridization with FGF4 probe was analyzed using tissue microarray consisting of representative cores of 267 ESCC cases. FGF4 amplification was observed in 52.8% (141/267) of patients. Patients with FGF4 amplification showed a significantly shorter disease-free survival (DFS) or disease-specific overall survival (OS) compared with those without FGF4 amplification (both P < .05). Moreover, FGF4 amplification was an independent prognostic factor (DFS, P = .036; OS, P = .021) along with clinical stage and lymph node metastasis in multivariate analysis. Among stage I-II or III patients whose DFS was greater than or equal to 24 months (n = 125 or 32), patients with FGF4 amplification showed a significantly worse prognosis (OS, P = .027 or P = .010). Moreover, the survival curve of stage I-II patients with FGF4 amplification was identical to stage III patients without FGF4 amplification (DFS, P = .643; OS, P = .707). Taken together, FGF4 amplification was an independent prognostic factor in ESCC patients, and ESCC might have potentially been upstaged by FGF4 amplification. Therefore, FGF4 amplification in combination with clinical stage could be used as a relatively accurate predictor for the 5-year probability of death and recurrence for ESCC patients.

Objectives The boundary where inner and outer enamel epithelia meet is referred to as the cervical loop (CL) in molar tooth germs. In contrast, rodent incisors are continuously growing: the labial side of the teeth is covered with enamel (crown-analog), and the lingual side is covered with cementum (root-analog). These results in the appearance of CL in the frontal sections apart from the apical end. However, many researchers have used the term “labial CL” to indicate the apical end of incisors. Design This study investigated the gene expression patterns for the enamel knot signaling center in tooth morphogenesis to clarify the difference between “labial CL” and molar CL. We examined the three-dimensional expression patterns of markers for the enamel knot including fibroblast growth factor 4 (Fgf4), sonic hedgehog (Shh), Msx2, and P21 in frontal sections of murine mandibular incisors. Results Serial frontal sections from the apical end of the postnatal incisor clearly demonstrated the existence of enamel knot-like structures composed of supra-inner enamel epithelium and stellate reticulum in the “labial CL”. This structure includes the expression of all examined markers for enamel knots such as Fgf4, Shh, Msx2, and P21. Conclusions The molar tooth germ-like structure is maintained indefinitely in the “labial CL”. Therefore, the “labial CL” is not equivalent to the molar CL. The existence of an EK-like structure in the apical end of incisors implies that the usage of “labial CL” may be inappropriate for indicating this region. The “apical bud” could be used as an alternative term.

Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study, we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extraembryonic endoderm stem (iXEN) cells. Intriguingly, Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore, GATA6 induction in human embryonic stem (hES) cells also down-regulates pluripotency gene expression and up-regulates extraembryonic endoderm (ExEn) genes, revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement, with initial repression of Nanog and Esrrb, then Sox2, and finally Oct4, alongside step-wise activation of ExEn genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near pluripotency and endoderm genes, suggesting that Gata6 functions as both a direct repressor and activator. Together, this demonstrates that Gata6 is a versatile and potent reprograming factor that can act alone to drive a cell fate switch from diverse cell types.

Background/aim We report the outcomes of sorafenib therapy for advanced hepatocellular carcinoma (HCC) in our Department. Patients and methods Thirty-eight patients with unresectable HCC who were administrated sorafenib from 2009 to 2015 were investigated retrospectively. Results The 1-year overall survival rate was 59.3%. The macroscopic vascular invasion and response rate were independent prognostic factors of survival. Surgical resection after sorafenib achieved long-term survival in two cases. Case 1: A patient with locally unresectable HCC showed significant response induced by sorafenib, which allowed complete surgical resection. This tumor tested positive for FGF4. Case 2: A patient with a history of hepatectomy for HCC had multiple distant metastases. Most lesions were reduced in size after sorafenib therapy and new lesions in the remnant liver and residual lung metastases were resected. The sorafenib-resistant lesions were negative for FGF4. Conclusion Sorafenib combined with surgical resection is a feasible option in advanced HCC patients, if sorafenib has been effective.

Significance Chondrodystrophy, characterized by short limbs and intervertebral disc disease (IVDD), is a common phenotype in many of the most popular dog breeds, including the dachshund, beagle, and French bulldog. Here, we report the identification of a FGF4 retrogene insertion on chromosome 12, the second FGF4 retrogene reported in the dog, as responsible for chondrodystrophy and IVDD. Identification of the causative mutation for IVDD will impact an incredibly large proportion of the dog population and provides a model for IVDD in humans, as FGF-associated mutations are responsible for IVDD and short stature in human achondroplasia. This is a report of a second retrogene copy of the same parental gene, each causing complementary disease phenotypes in a mammalian species., Chondrodystrophy in dogs is defined by dysplastic, shortened long bones and premature degeneration and calcification of intervertebral discs. Independent genome-wide association analyses for skeletal dysplasia (short limbs) within a single breed (PBonferroni = 0.01) and intervertebral disc disease (IVDD) across breeds (PBonferroni = 4.0 × 10−10) both identified a significant association to the same region on CFA12. Whole genome sequencing identified a highly expressed FGF4 retrogene within this shared region. The FGF4 retrogene segregated with limb length and had an odds ratio of 51.23 (95% CI = 46.69, 56.20) for IVDD. Long bone length in dogs is a unique example of multiple disease-causing retrocopies of the same parental gene in a mammalian species. FGF signaling abnormalities have been associated with skeletal dysplasia in humans, and our findings present opportunities for both selective elimination of a medically and financially devastating disease in dogs and further understanding of the ever-growing complexity of retrogene biology.

Oct4 and Sox2 regulate the expression of target genes such as Nanog, Fgf4, and Utf1, by binding to their respective regulatory motifs. Their functional cooperation is reflected in their ability to heterodimerize on adjacent cis regulatory motifs, the composite Sox/Oct motif. Given that Oct4 and Sox2 regulate many developmental genes, a quantitative analysis of their synergistic action on different Sox/Oct motifs would yield valuable insights into the mechanisms of early embryonic development. In the present study, we measured binding affinities of Oct4 and Sox2 to different Sox/Oct motifs using fluorescence correlation spectroscopy. We found that the synergistic binding interaction is driven mainly by the level of Sox2 in the case of the Fgf4 Sox/Oct motif. Taking into account Sox2 expression levels fluctuate more than Oct4, our finding provides an explanation on how Sox2 controls the segregation of the epiblast and primitive endoderm populations within the inner cell mass of the developing rodent blastocyst.

Summary In morphological terms, “form” is used to describe an object’s shape and size. In dogs, facial form is stunningly diverse. Facial retrusion, the proximodistal shortening of the snout and widening of the hard palate is common to brachycephalic dogs and is a welfare concern, as the incidence of respiratory distress and ocular trauma observed in this class of dogs is highly correlated with their skull form. Progress to identify the molecular underpinnings of facial retrusion is limited to association of a missense mutation in BMP3 among small brachycephalic dogs. Here, we used morphometrics of skull isosurfaces derived from 374 pedigree and mixed-breed dogs to dissect the genetics of skull form. Through deconvolution of facial forms, we identified quantitative trait loci that are responsible for canine facial shapes and sizes. Our novel insights include recognition that the FGF4 retrogene insertion, previously associated with appendicular chondrodysplasia, also reduces neurocranium size. Focusing on facial shape, we resolved a quantitative trait locus on canine chromosome 1 to a 188-kb critical interval that encompasses SMOC2. An intronic, transposable element within SMOC2 promotes the utilization of cryptic splice sites, causing its incorporation into transcripts, and drastically reduces SMOC2 gene expression in brachycephalic dogs. SMOC2 disruption affects the facial skeleton in a dose-dependent manner. The size effects of the associated SMOC2 haplotype are profound, accounting for 36% of facial length variation in the dogs we tested. Our data bring new focus to SMOC2 by highlighting its clinical implications in both human and veterinary medicine., Highlights • A population-based genetics study of dogs that required diagnostic imaging • Resolution of a QTL associated with face length reduction (brachycephaly) • Association of brachycephaly with a retrotransposon that disrupts SMOC2 splicing • The SMOC2 locus explains up to 36% of face length variation in dogs, Uncovering the molecular basis of dogs’ vastly diverse skull shapes requires individualized approaches to morphometrics and genotyping. Using data from veterinary referral patients, Marchant et al. shed light on a large effect locus that is responsible for face length reduction (brachycephaly).

Glucose metabolism in trophoblast cells is essential to provide the required energy for the development and function of the placenta. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh), a key enzyme in the glycolysis pathway has been considered ubiquitously expressed in cells. There is, however, a growing body of evidence suggesting that Gapdh has many functions in pathways unrelated to glucose metabolism. In the present study, we show that GAPDH expression and sub-cellular localization changes through gestation in the mouse placenta. Our findings raise the possibility that GAPDH has multiple functions in trophoblast cells and the developing placenta, while also cautioning against its use as an endogenous reference or standard for gene expression in the placenta.

Article, Cardiovascular diseases remain a large global health problem. Although several conventional small-molecule treatments are available for common cardiovascular problems, gene therapy is a potential treatment option for acquired and inherited cardiovascular diseases that remain with unmet clinical needs. Among potential targets for gene therapy are severe cardiac and peripheral ischemia, heart failure, vein graft failure, and some forms of dyslipidemias. The first approved gene therapy in the Western world was indicated for lipoprotein lipase deficiency, which causes high plasma triglyceride levels. With improved gene delivery methods and more efficient vectors, together with interventional transgene strategies aligned for a better understanding of the pathophysiology of these diseases, new approaches are currently tested for safety and efficacy in clinical trials. In this article, we integrate a historical perspective with recent advances that will likely affect clinical development in this research area., final draft, http://purl.org/eprint/status/PeerReviewed

The first electrochemical immunosensor for the determination of fibroblast growth factor receptor 4 (FGFR4) biomarker is reported in this work. The biosensor involves a sandwich configuration with covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic microcarriers (HOOC-MBs) and amperometric detection at disposable carbon screen-printed electrodes (SPCEs). The biosensor exhibits a great analytical performance regarding selectivity for the target protein and a low LOD of 48.2 pg mL-1. The electrochemical platform was successfully applied for the determination of FGFR4 in different cancer cell lysates without any apparent matrix effect after a simple sample dilution and using only 2.5 μg of the raw lysate. Comparison of the results with those provided by a commercial ELISA kit shows competitive advantages by using the developed immunosensor in terms of simplicity, analysis time, and portability and cost-affordability of the required instrumentation for the accurate determination of FGFR4 in cell lysates.

The development of the craniofacial muscles requires reciprocal interactions with surrounding craniofacial tissues that originate from cranial neural crest cells (CNCCs). However, the molecular mechanism involved in the tissue-tissue interactions between CNCCs and muscle progenitors during craniofacial muscle development is largely unknown. In the current study, we address how CNCCs regulate the development of the tongue and other craniofacial muscles using Wnt1-Cre; Alk5(fl/fl) mice, in which loss of Alk5 in CNCCs results in severely disrupted muscle formation. We found that Bmp4 is responsible for reduced proliferation of the myogenic progenitor cells in Wnt1-Cre; Alk5(fl/fl) mice during early myogenesis. In addition, Fgf4 and Fgf6 ligands were reduced in Wnt1-Cre; Alk5(fl/fl) mice and are critical for differentiation of the myogenic cells. Addition of Bmp4 or Fgf ligands rescues the proliferation and differentiation defects in the craniofacial muscles of Alk5 mutant mice in vitro. Taken together, our results indicate that CNCCs play critical roles in controlling craniofacial myogenic proliferation and differentiation through tissue-tissue interactions.

Summary Cell signaling depends on dynamic protein-protein interaction (PPI) networks, often assembled through modular domains each interacting with multiple peptide motifs. This complexity raises a conceptual challenge, namely to define whether a particular cellular response requires assembly of the complete PPI network of interest or can be driven by a specific interaction. To address this issue, we designed variants of the Grb2 SH2 domain ("pY-clamps") whose specificity is highly biased toward a single phosphotyrosine (pY) motif among many potential pYXNX Grb2-binding sites. Surprisingly, directing Grb2 predominantly to a single pY site of the Ptpn11/Shp2 phosphatase, but not other sites tested, was sufficient for differentiation of the essential primitive endoderm lineage from embryonic stem cells. Our data suggest that discrete connections within complex PPI networks can underpin regulation of particular biological events. We propose that this directed wiring approach will be of general utility in functionally annotating specific PPIs.

Cells of the inner cell mass (ICM) of the mouse blastocyst differentiate into the pluripotent epiblast (EPI) or the primitive endoderm (PrE), marked by the transcription factors NANOG and GATA6, respectively. To investigate the mechanistic regulation of this process, we applied an unbiased, quantitative, single-cell resolution image analysis pipeline, to analyze embryos lacking or exhibiting reduced levels of GATA6. We find that Gata6 mutants exhibit a complete absence of PrE, and demonstrate that GATA6 levels regulate the timing and speed of lineage commitment within the ICM. Furthermore, we show that GATA6 is necessary for PrE specification by FGF signaling, and propose a model where interactions between NANOG, GATA6 and the FGF/ERK pathway determine ICM cell fate. This study provides a framework for quantitative analyses of mammalian embryos, and establishes GATA6 as a nodal point in the gene regulatory network driving ICM lineage specification.

The aim of this study was to investigate whether the modification of bone marrow-derived mesenchymal stem cells (BMSCs) with the fused FGF4 (fibroblast growth factor 4)-bFGF (basic fibroblast growth factor) gene could improve the expression and secretion of BFGF, and increase the efficacies in repairing infarcted myocardium. We used In-Fusion technique to construct recombinant lentiviral vectors containing the individual gene of bFGF, enhanced green fluorescent protein (EGFP), or genes of FGF4-bFGF and EGFP, and then transfected these lentiviruses into rat BMSCs. We conducted an in vitro experiment to compare the secretion of bFGF in BMSCs infected by these lentiviruses and also examined their therapeutic effects in the treatment of myocardial infraction in a rodent study. Sixty rats were tested in the following five conditions: Group-SHAM received only sham operation as controls; Group-AMI received only injection of placebo PBS buffer; Group-BMSC, Group-bFGF and Group-FGF4-bFGF received implantation of BMSCs with empty lentivirus, bFGF lentivirus, and FGF4-bFGF lentivirus, respectively. Our results found out that the transplanted FGF4-bFGF BMSCs had the highest survival rate, and also the highest myocardial expression of bFGF and microvascular density as evidenced by Western blotting and immunohistochemistry, respectively. As compared to other groups, the Group-FGF4-BFGF rats had the lowest myocardial fibrotic fraction, and the highest left ventricular ejection fraction. These results suggest that the modification of BMSCs with the FGF4-bFGF fused gene can not only increase the expression of bFGF but also improve its secretion. The FGF4-bFGF BMSCs thus can enhance the survival of the transplanted cells, diminish myocardial fibrosis, promote myocardial angiogenesis, and improve cardiac functions.

Here, we generated a genome-scale shRNA library targeting long intergenic noncoding RNAs (lincRNAs) in the mouse. We performed an unbiased loss-of-function study in mouse embryonic stem cells (mESCs) and identified 20 lincRNAs involved in the maintenance of pluripotency. Among these, TUNA (Tcl1 Upstream Neuron-Associated lincRNA, or megamind) was required for pluripotency and formed a complex with three RNA-binding proteins (RBPs). The TUNA-RBP complex was detected at the promoters of Nanog, Sox2, and Fgf4, and knockdown of TUNA or the individual RBPs inhibited neural differentiation of mESCs. TUNA showed striking evolutionary conservation of both sequence- and CNS-restricted expression in vertebrates. Accordingly, knockdown of tuna in zebrafish caused impaired locomotor function, and TUNA expression in the brains of Huntington's disease patients was significantly associated with disease grade. Our results suggest that the lincRNA TUNA plays a vital role in pluripotency and neural differentiation of ESCs and is associated with neurological function of adult vertebrates.

Mineral trioxide aggregate (MTA) and calcium-enriched mixture (CEM) have shown osteogenic/cementogenic/dentinogenic activities; however, their mechanism of action is not fully understood. We aimed to evaluate the effect of these biomaterials on odontogenic differentiation of human dental pulp stem cells (DPSCs).Flow cytometry with stem cell markers for the confirmation of stemness and homogeneity was first performed. Then isolated DPSCs were seeded on prepared discs of MTA, CEM, differentiation medium (DM), and growth medium (GM) and incubated up to 14 days. Concentrations of transforming growth factor-β1, bone morphogenetic protein (BMP)2, BMP4, and fibroblast growth factor 4 were measured at each interval using an enzyme-linked immunosorbent assay reader. Gene expression of dentin sialophosphoprotein, dentin matrix protein 1, and the cytokines were evaluated by reverse-transcription polymerase chain reaction. To evaluate the cell morphology, scanning electron micrographs were taken; mineralization potential was evaluated using alizarin red S staining.Scanning electron micrographs showed that DPSCs spread/adhered/proliferated similarly on MTA and CEM. On day 14, alizarin red S staining confirmed that mineralization occurred in all groups except GM. Expressions of dentin matrix protein 1 and dentin sialophosphoprotein genes were similar in the CEM, MTA, and DM groups; they were significantly higher compared with the GM group (P.05). A greater amount of transforming growth factor-β1 gene was expressed in MTA compared with the other groups (P.05). However, the expression of fibroblast growth factor 4 and BMP2 genes was significantly greater in the CEM group (P.05). In all the tested groups, the expression of BMP4 was less than GM (P.01); however, CEM and DM were similar but more than MTA (P.05). Concentrations of protein product detected using an enzyme-linked immunosorbent assay reader confirmed these gene expressions.MTA and CEM can induce osteo-/odontogenic-like phenotype differentiation of human DPSCs; however, they stimulate different gene expressions and growth factor release.

The crosstalk between the epiblast and the trophoblast is critical in supporting the early stages of conceptus development. FGF4 and BMP4 are inductive signals that participate in the communication between the epiblast and the extraembryonic ectoderm (ExE) of the developing mouse embryo. Importantly, however, it is unknown whether a similar crosstalk operates in species that lack a discernible ExE and develop a mammotypical embryonic disc (ED). Here we investigated the crosstalk between the epiblast and the trophectoderm (TE) during pig embryo elongation. FGF4 ligand and FGFR2 were detected primarily on the plasma membrane of TE cells of peri-elongation embryos. The binding of this growth factor to its receptor triggered a signal transduction response evidenced by an increase in phosphorylated MAPK/ERK. Particular enrichment was detected in the periphery of the ED in early ovoid embryos, indicating that active FGF signalling was operating during this stage. Gene expression analysis shows that CDX2 and ELF5, two genes expressed in the mouse ExE, are only co-expressed in the Rauber's layer, but not in the pig mural TE. Interestingly, these genes were detected in the nascent mesoderm of early gastrulating embryos. Analysis of BMP4 expression by in situ hybridisation shows that this growth factor is produced by nascent mesoderm cells. A functional test in differentiating epiblast shows that CDX2 and ELF5 are activated in response to BMP4. Furthermore, the effects of BMP4 were also demonstrated in the neighbouring TE cells, as demonstrated by an increase in phosphorylated SMAD1/5/8. These results show that BMP4 produced in the extraembryonic mesoderm is directly influencing the SMAD response in the TE of elongating embryos. These results demonstrate that paracrine signals from the embryo, represented by FGF4 and BMP4, induce a response in the TE prior to the extensive elongation. The study also confirms that expression of CDX2 and ELF5 is not conserved in the mural TE, indicating that although the signals that coordinate conceptus growth are similar between rodents and pigs, the gene regulatory network of the trophoblast lineage is not conserved in these species.

Our recent studies demonstrated that mature astrocytes from spinal cord can be reprogrammed in vitro and in vivo to generate neural stem/progenitor cells (NSPCs) following treatment with conditioned medium collected from mechanically injured astrocytes. However, little is known regarding the molecular mechanisms underlying the reprogramming of astrocytes. Here, we show that fibroblast growth factor 4 (FGF4) exerts a critical role in synergistically converting astrocytes into NSPCs that can express multiple neural stem cell markers (nestin and CD133) and are capable of both self-renewal and differentiation into neurons and glia. Lack of FGF4 signals fails to elicit the dedifferentiation of astrocytes towards NSPCs, displaying a substantially lower efficiency in the reprogramming of astrocytes and a slower transition through fate-determined state. These astrocyte-derived NSPCs displayed relatively poor self-renewal and multipotency. More importantly, further investigation suggested that FGF4 is a key molecule necessary for activating PI3K/Akt/p21 signaling cascades, as well as their downstream effectors responsible for directing cell reprogramming towards NSPCs. Collectively, these findings provide a molecular basis for astrocyte dedifferentiation into NSPCs after central nervous system (CNS) injury and imply that FGF4 may be a clinically applicable molecule for in situ neural repair in the CNS disorders.

Mesenchymal stem cells (MSCs) are an active topic of research in regenerative medicine due to their ability to secrete a variety of growth factors and cytokines that promote healing of damaged tissues and organs. In addition, these secreted growth factors and cytokines have been shown to exert an autocrine effect by regulating MSC proliferation and differentiation. We found that expression of EGF, FGF-4 and HGF were down-regulated during serial passage of bone marrow-derived mesenchymal stem cells (BMSCs). Proliferation and differentiation potentials of BMSCs treated with these growth factors for 2 months were evaluated and compared to BMSCs treated with FGF-2, which increased proliferation of BMSCs. FGF-2 and -4 increased proliferation potentials at high levels, about 76- and 26-fold, respectively, for 2 months, while EGF and HGF increased proliferation of BMSCs by less than 2.8-fold. Interestingly, differentiation potential, especially adipogenesis, was maintained only by HGF treatment. Treatment with FGF-2 rapidly induced activation of AKT and later induced ERK activation. The basal level of phosphorylated ERK increased during serial passage of BMSCs treated with FGF-2. The expression of LC3-II, an autophagy marker, was gradually increased and the population of senescent cells was increased dramatically at passage 7 in non-treated controls. But FGF-2 and FGF-4 suppressed LC3-II expression and down-regulated senescent cells during long-term (i.e. 2month) cultures. Taken together, depletion of growth factors during serial passage could induce autophagy, senescence and down-regulation of stemness (proliferation via FGF-2/-4 and differentiation via HGF) through suppression of AKT and ERK signaling.

This study was undertaken to establish rat embryonic stem (ES) cells from parthenogenetically developing blastocysts. Ten blastocysts were prepared by treatment of ovulated rat oocytes with ionomycin and cycloheximide, and three alkaline phosphatase-positive ES cell lines were established using the N2B27 medium supplemented with mitogen activated protein kinase kinase inhibitor PD0325901, glycogen synthase kinase 3 inhibitor CHIR99021, rat leukemia inhibitory factor, and forskolin. Expression of stem cell marker genes (Oct-4, rNanog, Fgf-4, and Rex-1) was confirmed in all three ES cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR). Combined bisulfite restriction analysis showed that the differentially methylated region locus of five imprinted genes (H19, Meg3IG, Igf2r, Peg5, and Peg10) in these ES cells remained to be demethylated or was hypomethylated, which was similar to that in control ES cells established from normal blastocysts. Characteristics of the parthenogenetic blastocyst-derived ES cells were successfully transmitted to the next generation through a chimeric rat for one of the three ES cell lines. This is the first report on germline-competent (genuine) ES cells derived from parthenogenetically developing rat blastocysts.

The epiblast (EPI) and the primitive endoderm (PE), which constitute foundations for the future embryo body and yolk sac, build respectively deep and surface layers of the inner cell mass (ICM) of the blastocyst. Before reaching their target localization within the ICM, the PE and EPI precursor cells, which display distinct lineage-specific markers, are intermingled randomly. Since the ICM cells are produced in two successive rounds of asymmetric divisions at the 8→16 (primary inner cells) and 16→32 cell stage (secondary inner cells) it has been suggested that the fate of inner cells (decision to become EPI or PE) may depend on the time of their origin. Our method of dual labeling of embryos allowed us to distinguish between primary and secondary inner cells contributing ultimately to ICM. Our results show that the presence of two generations of inner cells in the 32-cell stage embryo is the source of heterogeneity within the ICM. We found some bias concerning the level of Fgf4 and Fgfr2 expression between primary and secondary inner cells, resulting from the distinct number of cells expressing these genes. Analysis of experimental aggregates constructed using different ratios of inner cells surrounded by outer cells revealed that the fate of cells does not depend exclusively on the timing of their generation, but also on the number of cells generated in each wave of asymmetric division. Taking together, the observed regulatory mechanism adjusting the proportion of outer to inner cells within the embryo may be mediated by FGF signaling.

Summary: Trophoblast stem (TS) cells are increasingly used as a model system for studying placentation and placental disorders. However, practical limitations of genetic manipulation have posed challenges for genetic analysis using TS cells. Here, we report the generation of mouse parthenogenetic haploid TS cells (haTSCs) and show that supplementation with FGF4 and inhibition of Rho-associated protein kinase (ROCK) enable the maintenance of their haploidy and developmental potential. The resulting haTSCs have 20 chromosomes, exhibit typical expression features of TS cells, possess the multipotency to differentiate into specialized trophoblast cell types, and can chimerize E13.5 and term placentas. We also demonstrate the capability of the haTSCs to undergo genetic manipulation and facilitate genome-wide screening in the trophoblast lineage. We expect that haTSCs will offer a powerful tool for studying functional genomics and placental biology. : Trophoblast stem (TS) cells are increasingly used as a model system for studying placentation and placental disorders. Here, Cui et al. report the generation of mouse haploid TS cells, which possess a wide extraembryonic developmental potential and can serve as a powerful tool for studying functional genomics and placental biology. Keywords: haploidy, trophoblast, stem cells, TSC

The primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Although it has been shown that FGF signaling is necessary and sufficient for PE specification in the ICM, it is unknown what mechanisms control the PE/EPI proportion in the embryo. Because modulation of FGF signaling alone is sufficient to convert all ICM cells to either PE or EPI, a model has been proposed in which the amount of FGF in the embryo controls the PE/EPI proportion. To test this model, we reduced the amount of FGF4, the major FGF in the preimplantation embryo, using various genotypes of Fgf4 mutants. We observed a maternal contribution of Fgf4 in PE specification, but it was dispensable for development. In addition, upon treatment of Fgf4 mutant embryos with exogenous FGF4, we observed a progressive increase of PE proportions in an FGF4 dose dependent manner, regardless of embryo genotype. We conclude that the amount of FGF4 is limited and regulates PE/EPI proportions in the mouse embryo.