Requirement for STAT3 and its target, TFCP2L1, in self-renewal of naïve pluripotent stem cells in vivo and in vitro.

We previously demonstrated gradual loss of epiblast during diapause in embryos lacking components of the LIF/IL6 receptor. Here, we explore the requirement for the downstream signalling transducer andactivator of transcription STAT3 and its target, TFCP2L1, in maintenance of naïve pluripotency. Unlike conventional markers, such as NANOG, which remains high in epiblast until implantation, both STAT3 and TFCP2L1 proteins decline during blastocyst expansion, but intensify in the embryonic region after induction of diapause, as observed visually and confirmed using our image-analysis pipeline, consistent with our previous transcriptional expression data. Embryos lacking STAT3 or TFCP2L1 underwent catastrophic loss of most of the inner cell mass during the first few days of diapause, indicating involvement of signals in addition to LIF/IL6 for sustaining naïve pluripotency in vivo. By blocking MEK/ERK signalling from the morula stage, we could derive embryonic stem cells with high efficiency from STAT3 null embryos, but not those lacking TFCP2L1, suggesting a hitherto unknown additional role for this essential STAT3 target in transition from embryo to embryonic stem cells in vitro. This article has an associated First Person interview with the first author of the paper.

Essential Roles of Exocyst Complex Component 3-like 2 on Cardiovascular Development in Mice.

Angiogenesis is a process to generate new blood vessels from pre-existing vessels and to maintain vessels, and plays critical roles in normal development and disease. However, the molecular mechanisms underlying angiogenesis are not fully understood. This study examined the roles of exocyst complex component (Exoc) 3-like 2 (Exoc3l2) during development in mice. We found that Exoc3l1, Exoc3l2, Exoc3l3 and Exoc3l4 are expressed abundantly in endothelial cells at embryonic day 8.5. The generation of Exoc3l2 knock-out (KO) mice showed that disruption of Exoc3l2 resulted in lethal in utero. Substantial numbers of Exoc3l2 KO embryos exhibited hemorrhaging. Deletion of Exoc3l2 using Tie2-Cre transgenic mice demonstrated that Exoc3l2 in hematopoietic and endothelial lineages was responsible for the phenotype. Taken together, these findings reveal that Exoc3l2 is essential for cardiovascular and brain development in mice.

Regulation of the ERK signalling pathway in the developing mouse blastocyst.

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 occurs 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. Whereas 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 the PrE while protecting the Epi identity from autocrine signalling.

Generation of bicistronic reporter knockin mice for visualizing germ layers.

Knockout mouse models are commonly used in developmental biology to investigate the functions of specific genes, and the knowledge obtained in such models has yielded insights into the molecular mechanisms underlying developmental processes. Gastrulation is the most dynamic process in embryogenesis during which differentiation into three germ layers occurs. However, the functions of genes involved in gastrulation are not completely understood. One major reason for this is the technical difficulty of embryo analysis to understand germ layer location. We have generated three reporter mouse strains in which the germ layers are distinguished by different fluorescent reporters. Using CRISPR/Cas9 genome editing in mouse zygotes, the fluorescent reporter genes, EGFP, tdTomato, and TagBFP including 2A peptide sequences were knocked into the appropriate sites before the stop codon of the Sox17 (endoderm marker), Otx2 (ectoderm marker), and T (mesoderm marker) genes, respectively. Founder mice were successfully generated in the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter strains. Further, homozygous knockin mice of all strains appeared morphologically normal and were fertile. On stereomicroscopic analysis, fluorescent signals were detected in a germ layer-specific manner from heterozygous embryos at embryonic day (E) 6.5-8.5 in all strains, and were immunohistochemically demonstrated to match their respective germ layer-specific marker protein at E7.5. Taken together, these observations suggest that the Sox17-2A-EGFP, Otx2-2A-tdTomato, and T-2A-TagBFP knockin reporter mice may be useful for comprehensive analysis of gene function in germ layer formation.

Comprehensive evaluation of ubiquitous promoters suitable for the generation of transgenic cynomolgus monkeys†.

Nonhuman primates (NHPs) are considered to be the most valuable models for human transgenic (Tg) research into disease because human pathology is more closely recapitulated in NHPs than rodents. Previous studies have reported the generation of Tg NHPs that ubiquitously overexpress a transgene using various promoters, but it is not yet clear which promoter is most suitable for the generation of NHPs overexpressing a transgene ubiquitously and persistently in various tissues. To clarify this issue, we evaluated four putative ubiquitous promoters, cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin (CAG), elongation factor 1α (EF1α), ubiquitin C (UbC), and CMV, using an in vitro differentiation system of cynomolgus monkey embryonic stem cells (ESCs). While the EF1α promoter drove Tg expression more strongly than the other promoters in undifferentiated pluripotent ESCs, the CAG promoter was more effective in differentiated cells such as embryoid bodies and ESC-derived neurons. When the CAG and EF1α promoters were used to generate green fluorescent protein (GFP)-expressing Tg monkeys, the CAG promoter drove GFP expression in skin and hematopoietic tissues more strongly than in ΕF1α-GFP Tg monkeys. Notably, the EF1α promoter underwent more silencing in both ESCs and Tg monkeys. Thus, the CAG promoter appears to be the most suitable for ubiquitous and stable expression of transgenes in the differentiated tissues of Tg cynomolgus monkeys and appropriate for the establishment of human disease models.

Generating Vegfr3 reporter transgenic mouse expressing membrane-tagged Venus for visualization of VEGFR3 expression in vascular and lymphatic endothelial cells.

Vascular endothelial growth factor receptor 3 (Vegfr3) has been widely used as a marker for lymphatic and vascular endothelial cells during mouse embryonic development and in adult mouse, making it valuable for studying angiogenesis and lymphangiogenesis under normal and pathological conditions. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a membrane-localized fluorescent reporter protein, Gap43-Venus, under the control of the Vegfr3 regulatory sequence. Vegfr3-Gap43-Venus BAC Tg recapitulated endogenous Vegfr3 expression in vascular and lymphatic endothelial cells during embryonic development and tumor development. Thus, this Tg mouse line contributes a valuable model to study angiogenesis and lymphangiogenesis in physiological and pathological contexts.

Competition for Mitogens Regulates Spermatogenic Stem Cell Homeostasis in an Open Niche.

In many tissues, homeostasis is maintained by physical contact between stem cells and an anatomically defined niche. However, how stem cell homeostasis is achieved in environments where cells are motile and dispersed among their progeny remains unknown. Using murine spermatogenesis as a model, we find that spermatogenic stem cell density is tightly regulated by the supply of fibroblast growth factors (FGFs) from lymphatic endothelial cells. We propose that stem cell homeostasis is achieved through competition for a limited supply of FGFs. We show that the quantitative dependence of stem cell density on FGF dosage, the biased localization of stem cells toward FGF sources, and stem cell dynamics during regeneration following injury can all be predicted and explained within the framework of a minimal theoretical model based on "mitogen competition." We propose that this model provides a generic and robust mechanism to support stem cell homeostasis in open, or facultative, niche environments.

Klf5 suppresses ERK signaling in mouse pluripotent stem cells.

Mouse embryonic stem cells (ESCs) are pluripotent stem cells, which have the ability to differentiate into all three germ layers: mesoderm, endoderm, and ectoderm. Proper levels of phosphorylated extracellular signal-regulated kinase (pERK) are critical for maintaining pluripotency, as elevated pERK evoked by fibroblast growth factor (FGF) receptor activation results in differentiation of ESCs, while, conversely, reduction of pERK by a MEK inhibitor maintains a pluripotent ground state. However, mechanisms underlying proper control of pERK levels in mouse ESCs are not fully understood. Here, we find that Klf5, a Krüppel-like transcription factor family member, is a component of pERK regulation in mouse ESCs. We show that ERK signaling is overactivated in Klf5-KO ESCs and the overactivated ERK in Klf5-KO ESCs is suppressed by the introduction of Klf5, but not Klf2 or Klf4, indicating a unique role for Klf5 in ERK suppression. Moreover, Klf5 regulates Spred1, a negative regulator of the FGF-ERK pathway. Klf5 also facilitates reprogramming of EpiSCs into a naïve state in combination with a glycogen synthase kinase 3 inhibitor and LIF, and in place of a MEK inhibitor. Taken together, these results show for the first time that Klf5 has a unique role suppressing ERK activity in mouse ESCs.

Klf5 maintains the balance of primitive endoderm versus epiblast specification during mouse embryonic development by suppression of Fgf4.

The inner cell mass of the mouse blastocyst gives rise to the pluripotent epiblast (EPI), which forms the embryo proper, and the primitive endoderm (PrE), which forms extra-embryonic yolk sac tissues. All inner cells coexpress lineage markers such as Nanog and Gata6 at embryonic day (E) 3.25, and the EPI and PrE precursor cells eventually segregate to exclusively express Nanog and Gata6, respectively. Fibroblast growth factor (FGF)-extracellular signal-regulated kinase (ERK) signalling is involved in segregation of the EPI and PrE lineages; however, the mechanism involved in Fgf4 regulation is poorly understood. Here, we identified Klf5 as an upstream repressor of Fgf4Fgf4 was markedly upregulated in Klf5 knockout (KO) embryos at E3.0, and was downregulated in embryos overexpressing Klf5 Furthermore, Klf5 KO and overexpressing blastocysts showed skewed lineage specification phenotypes, similar to FGF4-treated preimplantation embryos and Fgf4 KO embryos, respectively. Inhibitors of the FGF receptor (Fgfr) and ERK pathways reversed the skewed lineage specification of Klf5 KO blastocysts. These data demonstrate that Klf5 suppresses Fgf4-Fgfr-ERK signalling, thus preventing precocious activation of the PrE specification programme.

Visualization of the Epiblast and Visceral Endodermal Cells Using Fgf5-P2A-Venus BAC Transgenic Mice and Epiblast Stem Cells.

Fibroblast growth factor 5 (Fgf5) has been widely used as a marker for the epiblast in the postimplantation embryo and epiblast stem cells (mEpiSCs) in the mouse, making it valuable for study of differentiation of various tissues and epiblast cells in vivo and in vitro. Here, we report for the first time the generation of Fgf5-P2A-Venus BAC transgenic (Tg) mice and show that the BAC Tg can recapitulate endogenous Fgf5 expression in epiblast and visceral endodermal cells of E6.5 and 7.5 embryos. We also show that Fgf5-P2A-Venus BAC Tg mEpiSCs in the undifferentiated state expressed abundant Venus, and upon reprogramming into naïve state, Venus was suppressed. Furthermore, while most Tg mEpiSCs expressed Venus abundantly, surprisingly the Tg mEpiSCs contained a minor subpopulation of Venus-negative cells that were capable of conversion to Venus-positive cells, indicating that even Fgf5 expression shows dynamic heterogeneity in mEpiSCs. Taken together, Fgf5-P2A-Venus BAC Tg mice and mEpiSCs generated in this study will be useful for developmental biology as well as stem cell biology research.

Generation of transgenic cynomolgus monkeys that express green fluorescent protein throughout the whole body.

Nonhuman primates are valuable for human disease modelling, because rodents poorly recapitulate some human diseases such as Parkinson's disease and Alzheimer's disease amongst others. Here, we report for the first time, the generation of green fluorescent protein (GFP) transgenic cynomolgus monkeys by lentivirus infection. Our data show that the use of a human cytomegalovirus immediate-early enhancer and chicken beta actin promoter (CAG) directed the ubiquitous expression of the transgene in cynomolgus monkeys. We also found that injection into mature oocytes before fertilization achieved homogenous expression of GFP in each tissue, including the amnion, and fibroblasts, whereas injection into fertilized oocytes generated a transgenic cynomolgus monkey with mosaic GFP expression. Thus, the injection timing was important to create transgenic cynomolgus monkeys that expressed GFP homogenously in each of the various tissues. The strategy established in this work will be useful for the generation of transgenic cynomolgus monkeys for transplantation studies as well as biomedical research.

Comprehensive Identification of Krüppel-Like Factor Family Members Contributing to the Self-Renewal of Mouse Embryonic Stem Cells and Cellular Reprogramming.

Pluripotency is maintained in mouse embryonic stem (ES) cells and is induced from somatic cells by the activation of appropriate transcriptional regulatory networks. Krüppel-like factor gene family members, such as Klf2, Klf4 and Klf5, have important roles in maintaining the undifferentiated state of mouse ES cells as well as in cellular reprogramming, yet it is not known whether other Klf family members exert self-renewal and reprogramming functions when overexpressed. In this study, we examined whether overexpression of any representative Klf family member, such as Klf1-Klf10, would be sufficient for the self-renewal of mouse ES cells. We found that only Klf2, Klf4, and Klf5 produced leukemia inhibitory factor (LIF)-independent self-renewal, although most KLF proteins, if not all, have the ability to occupy the regulatory regions of Nanog, a critical Klf target gene. We also examined whether overexpression of any of Klf1-Klf10 would be sufficient to convert epiblast stem cells into a naïve pluripotent state and found that Klf5 had such reprogramming ability, in addition to Klf2 and Klf4. We also delineated the functional domains of the Klf2 protein for LIF-independent self-renewal and reprogramming. Interestingly, we found that both the N-terminal transcriptional activation and C-terminal zinc finger domains were indispensable for this activity. Taken together, our comprehensive analysis provides new insight into the contribution of Klf family members to mouse ES self-renewal and cellular reprogramming.

Generation of naive-like porcine-induced pluripotent stem cells capable of contributing to embryonic and fetal development.

In pluripotent stem cells (PSCs), there are 2 types: naive and primed. Only the naive type has the capacity for producing chimeric offspring. Mouse PSCs are naive, but human PSCs are in the primed state. Previously reported porcine PSCs appear in the primed state. In this study, putative naive porcine-induced pluripotent stem cells (iPSCs) were generated. Porcine embryonic fibroblasts were transduced with retroviral vectors expressing Yamanaka's 4 genes. Emergent colonies were propagated in the presence of porcine leukemia inhibitory factor (pLIF) and forskolin. The cells expressed pluripotency markers and formed embryoid bodies, which gave rise to cell types from all 3 embryonic germ layers. The naive state of the cells was demonstrated by pLIF dependency, 2 active X chromosomes (when female), absent MHC class I expression, and characteristic gene expression profiles. The porcine iPSCs contributed to the in vitro embryonic development (11/24, 45.8%) as assessed by fluorescent markers. They also contributed to the in utero fetal development (11/71, 15.5% at day 23; 1/13, 7.7% at day 65). This is the first demonstration of macroscopic fluorescent chimeras derived from naive-like porcine PSCs, although adult chimeras remain to be produced.

Study of normal and pathological blood vessel morphogenesis in Flt1-tdsRed BAC Tg mice.

Blood vessel development and network patterning are controlled by several signaling molecules, including VEGF, FGF, TGF-ß, and Ang-1,2. Among these, the role of VEGF-A signaling in vessel morphogenesis is best understood. The biological activity of VEGF-A depends on its reaction with specific receptors Flt1 and Flk1. Roles of VEGF-A signaling in endothelial cell proliferation, migration, survival, vascular permeability, and induction of tip cell filopodia have been reported. In this study, we have generated Flt1-tdsRed BAC transgenic (Tg) mice to monitor Flt1 gene expression during vascular development. We show that tdsRed fluorescence is observed within blood vessels of adult mice and embryos, indicative of retinal angiogenesis and tumor angiogenesis. Flt1 expression recapitulated by Flt1-tdsRed BAC Tg mice overlapped well with Flk1, while Flt1 was expressed more abundantly in endothelial cells of large blood vessels such as dorsal aorta and presumptive stalk cells in retina, providing a unique model to study blood vessel development.

Molecular basis for Flk1 expression in hemato-cardiovascular progenitors in the mouse.

The mouse Flk1 gene is expressed in various mesodermal progenitor cells of developing embryos. Recent studies have shown that Flk1 expression marks multipotent mesodermal progenitors, giving rise to various hemato-cardiovascular cell lineages during development. Flk1 expression also marks hemato-cardiovascular cell lineages in differentiating embryonic stem (ES) cells, which may be used in transplantation decisions to treat cardiovascular diseases. Despite its developmental and clinical importance in cardiovascular tissues, the transcriptional regulatory system of Flk1 has remained unclear. Here, we report a novel enhancer of the mouse Flk1 gene directing early mesodermal expression during development as well as ES differentiation. The enhancer enriches various mesodermal progenitors, such as primitive erythropoietic progenitors, hemangioblast (BL-CFC) and cardiovascular progenitors (CV-CFC). The enhancer is activated by Bmp, Wnt and Fgf, and it contains Gata-, Cdx-, Tcf/Lef-, ER71/Etv2- and Fox-binding sites, some of which are bound specifically by each of these transcription factors. As these transcription factors are known to act under the control of the Bmp, Wnt and Fgf families, early Flk1 expression may be induced by cooperative interactions between Gata, Tcf/Lef, Cdx and ER71/Etv2 under the control of Bmp, Wnt and Fgf signaling. The enhancer is required for early Flk1 expression and for hemangioblast development during ES differentiation.

Flk1-GFP BAC Tg mice: an animal model for the study of blood vessel development.

The mouse Flk1 (also called Kdr or Vegf-r2) gene encodes a receptor for VEGF-A. Flk1 is expressed in endothelial cells of the developing embryo. Recent studies have shown that Flk1 is expressed by multi-potent mesodermal progenitors, which give rise to various hematopoietic and cardiovascular cell lineages during development, and in differentiating ES cells, which may be used for cell transplantation therapy to treat cardiovascular diseases. Given its developmental and clinical importance in cardiovascular tissues, an animal model of Flk1 activity would be very useful. Here, we report the generation of Flk1-GFP BAC transgenic mice for monitoring Flk1 gene expression during development. We show that GFP expression in these mice serves as a surrogate marker for developing endothelial cells. Immunohistochemical analysis showed that the regions of expression of GFP and endogenous FLK1 largely overlap. Uniform GFP expression was observed in most endothelial cells at 8.5 dpc and thereafter. Flk1-GFP BAC transgenic mice should be useful for the study of both vascular development and pathological angiogenesis.