Gastrin-releasing peptide regulates fear learning under stressed conditions via activation of the amygdalostriatal transition area.

The amygdala, a critical brain region responsible for emotional behavior, is crucially involved in the regulation of the effects of stress on emotional behavior. In the mammalian forebrain, gastrin-releasing peptide (GRP), a 27-amino-acid mammalian neuropeptide, which is a homolog of the 14-amino-acid amidated amphibian peptide bombesin, is highly expressed in the amygdala. The levels of GRP are markedly increased in the amygdala after acute stress; therefore, it is known as a stress-activated modulator. To determine the role of GRP in emotional behavior under stress, we conducted some behavioral and biochemical experiments with GRP-knockout (KO) mice. GRP-KO mice exhibited a longer freezing response than wild-type (WT) littermates in both contextual and auditory fear (also known as threat) conditioning tests only when they were subjected to acute restraint stress 20 min before the conditioning. To identify the critical neural circuits associated with the regulation of emotional memory by GRP, we conducted Arc/Arg3.1-reporter mapping in the amygdala with an Arc-Venus reporter transgenic mouse line. In the amygdalostriatal transition area (AST) and the lateral side of the basal nuclei, fear conditioning after restraint stress increased neuronal activity significantly in WT mice, and GRP KO was found to negate this potentiation only in the AST. These results indicate that the GRP-activated neurons in the AST are likely to suppress excessive fear expression through the regulation of downstream circuits related to fear learning following acute stress.

CBP/p300 antagonises EGFR-Ras-Erk signalling and suppresses increased Ras-Erk signalling-induced tumour formation in mice.

CREB-binding protein (CBP) and p300 have oncogenic properties; both co-operate with pro-oncogenic transcription factors downstream of Ras-Erk signalling to support cell proliferation. By contrast, missense, truncating and in-frame mutations of CBP/p300 are found frequently in some human cancers, including cutaneous squamous cell carcinomas that originate from epidermal keratinocytes. Data support that dysfunction of CBP/p300 contributes to keratinocyte hyperproliferation and tumourigenesis; however, the mechanism by which dysfunction of CBP/p300 affects keratinocytes is unknown. Here, we used mice harbouring keratinocyte-specific genetic modifications to examine the role of CBP/p300 in the epidermis. While a single copy of either Crebbp or Ep300 was necessary and sufficient for maintaining epidermal development, reduced expression of CBP/p300 strengthened the Ras-Erk signalling-induced hyperplastic phenotype of epidermal keratinocytes. Reduced CBP/p300 expression increased ligand-induced EGFR activity while decreasing basal expression of Mig6, a negative regulator of EGFR. A reduction in CBP/p300, in combination with increased Ras-Erk signalling, also promoted epidermal tumour formation in mice. Thus, our findings support that CBP/p300 acts as a tumour suppressor in epidermal keratinocytes by counteracting EGFR-Ras-Erk signalling. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

FGF2-induced Ras-MAPK signalling maintains lymphatic endothelial cell identity by upregulating endothelial-cell-specific gene expression and suppressing TGFß signalling through Smad2.

The lymphatic endothelial cell (LEC) fate decision program during development has been described. However, the mechanism underlying the maintenance of differentiated LEC identity remains largely unknown. Here, we show that fibroblast growth factor 2 (FGF2) plays a fundamental role in maintaining a differentiated LEC trait. In addition to demonstrating the appearance of LECs expressing α-smooth muscle actin in mouse lymphedematous skin in vivo, we found that mouse immortalised LECs lose their characteristics and undergo endothelial-to-mesenchymal transition (EndMT) when cultured in FGF2-depleted medium. FGF2 depletion acted synergistically with transforming growth factor (TGF) ß to induce EndMT. We also found that H-Ras-overexpressing LECs were resistant to EndMT. Activation of H-Ras not only upregulated FGF2-induced activation of the Erk mitogen activated protein kinases (MAPK3 and MAPK1), but also suppressed TGFß-induced activation of Smad2 by modulating Smad2 phosphorylation by MAPKs. These results suggest that FGF2 regulates LEC-specific gene expression and suppresses TGFß signalling in LECs through Smad2 in a Ras-MAPK-dependent manner. Taken together, our findings provide a new insight into the FGF2-Ras-MAPK-dependent mechanism that maintains and modulates the LEC trait.

The Cd6 gene as a permissive locus for targeted transgenesis in the mouse.

The introduction of a transgene into the genome through homologous recombination or sequence-specific enzymatic modification is a key technique for producing transgenic mice. The Rosa26 gene has been widely used to produce transgenic mice because the gene is transcriptionally active in various cell types and, at many developmental stages, is permissive for constitutive expression of integrated transgenes, and is dispensable for normal development. However, permissive loci other than Rosa26 are needed to generate mice that harbor multiple transgenes for complex studies. Here, we identified the Cd6 locus on mouse chromosome 19 as a transgene integration site in a transgenic mouse strain showing widespread reporter expression. Using this locus, we generated a knock-in mouse line that harbors a CAG promoter-driven reporter transgene, and found that the homozygous transgenic mice are viable and fertile, although transgene insertion disrupted Cd6 gene expression. The transgene on the Cd6 locus expressed reporter genes extensively throughout embryos, neonates, and adults. Combined with the Cre/loxP binary system, blood and lymphatic endothelial cell-specific reporter expression from the transgenic locus was achieved. These results suggest that Cd6 is valuable as an alternative site for targeted transgenesis.

Development of a Mouse Experimental System for the In Vivo Characterization of Bioengineered Adipose-Derived Stromal Cells.

Human adipose-derived stromal cells (ADSCs) are an important resource for cell-based therapies. However, the dynamics of ADSCs after transplantation and their mechanisms of action in recipients remain unclear. Herein, we generated genetically engineered mouse ADSCs to clarify their biodistribution and post-transplantation status and to analyze their role in recipient mesenchymal tissue modeling. Immortalized ADSCs (iADSCs) retained ADSC characteristics such as stromal marker gene expression and differentiation potential. iADSCs expressing a fluorescent reporter gene were seeded into biocompatible nonwoven fabric sheets and transplanted into the dorsal subcutaneous region of neonatal mice. Transplanted donor ADSCs were distributed as CD90-positive stromal cells on the sheets and survived 1 month after transplantation. Although accumulation of T lymphocytes or macrophages inside the sheet was not observed with or without donor cells, earlier migration and accumulation of recipient blood vascular endothelial cells (ECs) inside the sheet was observed in the presence of donor cells. Thus, our mouse model can help in studying the interplay between donor ADSCs and recipient cells over a 1-month period. This system may be of value for assessing and screening bioengineered ADSCs in vivo for optimal cell-based therapies.

H-, N- and Kras cooperatively regulate lymphatic vessel growth by modulating VEGFR3 expression in lymphatic endothelial cells in mice.

Mammalian Ras, which is encoded by three independent genes, has been thought to be a versatile component of intracellular signalling. However, when, where and how Ras signalling plays essential roles in development and whether the three Ras genes have overlapping functions in particular cells remain unclear. Here, we show that the three Ras proteins dose-dependently regulate lymphatic vessel growth in mice. We find that lymphatic vessel hypoplasia is a common phenotype in Ras compound knockout mice and that overexpressed normal Ras in an endothelial cell lineage selectively causes lymphatic vessel hyperplasia in vivo. Overexpression of normal Ras in lymphatic endothelial cells leads to sustained MAPK activation, cellular viability and enhanced endothelial network formation under serum-depleted culture conditions in vitro, and knockdown of endogenous Ras in lymphatic endothelial cells impairs cell proliferation, MAPK activation, cell migration and endothelial network formation. Ras overexpression and knockdown result in up- and downregulation of vascular endothelial growth factor receptor (VEGFR) 3 expression, respectively, in lymphatic endothelial cells in vitro. The close link between Ras and VEGFR3 in vitro is consistent with the result that Ras knockout and transgenic alleles are genetic modifiers in lymphatic vessel hypoplasia caused by Vegfr3 haploinsufficiency. Our findings demonstrate a cooperative function of the three Ras proteins in normal development, and also provide a novel aspect of VEGFR3 signalling modulated by Ras in lymphangiogenesis.

An FGF4-FRS2alpha-Cdx2 axis in trophoblast stem cells induces Bmp4 to regulate proper growth of early mouse embryos.

A variety of stem cells are controlled by the actions of multiple growth factors in vitro. However, it remains largely unclear how growth factors control the proliferation and differentiation of stem cells in vivo. Here, we describe a novel paracrine mechanism for regulating a stem cell niche in early mammalian embryos, which involves communication between the inner cell mass (ICM) and the trophectoderm, from which embryonic stem (ES) cells and trophoblast stem (TS) cells can be derived, respectively. It is known that ES cells produce fibroblast growth factor (FGF)4 and that TS cells produce bone morphogenetic protein (Bmp)4. We provide evidence that FRS2alpha mediates activation of the extracellular signal-regulated progein kinase (ERK) pathway to enhance expression of transcription factor Cdx2 in TS cells in response to FGF4. Cdx2 in turn binds to an FGF4-responsive enhancer element of the promoter region of Bmp4, leading to production and secretion of Bmp4. Moreover, exogenous Bmp4 is able to rescue the defective growth of Frs2alpha-null ICM. These findings suggest an important role of Cdx2 for production of Bmp4 in TS cells to promote the proper growth of early mouse embryos.

Development of a new method for isolation and long-term culture of organ-specific blood vascular and lymphatic endothelial cells of the mouse.

Endothelial cells are indispensable components of the vascular system, and play pivotal roles during development and in health and disease. Their properties have been studied extensively by in vivo analysis of genetically modified mice. However, further analysis of the molecular and cellular phenotypes of endothelial cells and their heterogeneity at various developmental stages, in vascular beds and in various organs has often been hampered by difficulties in culturing mouse endothelial cells. In order to overcome these difficulties, we developed a new transgenic mouse line expressing the SV40 tsA58 large T antigen (tsA58T Ag) under the control of a binary expression system based on Cre/loxP recombination. tsA58T Ag-positive endothelial cells in primary cultures of a variety of organs proliferate continuously at 33 degrees C without undergoing cell senescence. The resulting cell population consists of blood vascular and lymphatic endothelial cells, which could be separated by immunosorting. Even when cultured for two months, the cells maintained endothelial cell properties, as assessed by expression of endothelium-specific markers and intracellular signaling through the vascular endothelial growth factor receptors VEGFR-2 and VEGFR-3, as well as their physiological characteristics. In addition, lymphatic vessel endothelial hyaluronan receptor-1 (Lyve-1) expression in liver sinusoidal endothelial cells in vivo was retained in vitro, suggesting that an organ-specific endothelial characteristic was maintained. These results show that our transgenic cell culture system is useful for culturing murine endothelial cells, and will provide an accessible method and applications for studying endothelial cell biology.

Phospholipase Cγ2 Is Required for Luminal Expansion of the Epididymal Duct during Postnatal Development in Mice.

Phospholipase Cγ2 (PLCγ2)-deficient mice exhibit misconnections of blood and lymphatic vessels, and male infertility. However, the cell type responsible for vascular partitioning and the mechanism for male infertility remain unknown. Accordingly, we generated a mouse line that conditionally expresses endogenous Plcg2 in a Cre/loxP recombination-dependent manner, and found that Tie2-Cre- or Pf4-Cre-driven reactivation of Plcg2 rescues PLCγ2-deficient mice from the vascular phenotype. By contrast, male mice rescued from the vascular phenotype exhibited epididymal sperm granulomas. As judged from immunostaining, PLCγ2 was expressed in clear cells in the epididymis. PLCγ2 deficiency did not compromise differentiation of epididymal epithelial cells, including clear cells, and tube formation at postnatal week 2. However, luminal expansion of the epididymal duct was impaired during the prepubertal period, regardless of epithelial cell polarity and tube architecture. These results suggest that PLCγ2-deficient clear cells cause impaired luminal expansion, stenosis of the epididymal duct, attenuation of luminal flow, and subsequent sperm granulomas. Clear cell-mediated luminal expansion is also supported by the observation that PLCγ2-deficient males were rescued from infertility by epididymal epithelium-specific reactivation of Plcg2, although the edematous and hemorrhagic phenotype associated with PLCγ2 deficiency also caused spontaneous epididymal sperm granulomas in aging males. Collectively, our findings demonstrate that PLCγ2 in clear cells plays an essential role in luminal expansion of the epididymis during the prepubertal period in mice, and reveal an unexpected link between PLCγ2, clear cells, and epididymal development.

Establishment of a tamoxifen-inducible Cre-driver mouse strain for widespread and temporal genetic modification in adult mice.

Temporal genetic modification of mice using the ligand-inducible Cre/loxP system is an important technique that allows the bypass of embryonic lethal phenotypes and access to adult phenotypes. In this study, we generated a tamoxifen-inducible Cre-driver mouse strain for the purpose of widespread and temporal Cre recombination. The new line, named CM32, expresses the GFPneo-fusion gene in a wide variety of tissues before FLP recombination and tamoxifen-inducible Cre after FLP recombination. Using FLP-recombined CM32 mice (CM32Δ mice) and Cre reporter mouse lines, we evaluated the efficiency of Cre recombination with and without tamoxifen administration to adult mice, and found tamoxifen-dependent induction of Cre recombination in a variety of adult tissues. In addition, we demonstrated that conditional activation of an oncogene could be achieved in adults using CM32Δ mice. CM32Δ;T26 mice, which harbored a Cre recombination-driven, SV40 large T antigen-expressing transgene, were viable and fertile. No overt phenotype was found in the mice up to 3 months after birth. Although they displayed pineoblastomas (pinealoblastomas) and/or thymic enlargement due to background Cre recombination by 6 months after birth, they developed epidermal hyperplasia when administered tamoxifen. Collectively, our results suggest that the CM32Δ transgenic mouse line can be applied to the assessment of adult phenotypes in mice with loxP-flanked transgenes.

Ras/MAPK signaling modulates VEGFR-3 expression through Ets-mediated p300 recruitment and histone acetylation on the Vegfr3 gene in lymphatic endothelial cells.

Modulation of VEGFR-3 expression is important for altering lymphatic endothelial cell (LEC) characteristics during the lymphangiogenic processes that occur under developmental, physiological, and pathological conditions. However, the mechanisms underlying the modulation of Vegfr3 gene expression remain largely unknown. Using genetically engineered mice and LECs, we demonstrated previously that Ras signaling is involved not only in VEGFR-3-induced signal transduction but also in Vegfr3 gene expression. Here, we investigated the roles of the transcription factor Ets and the histone acetyltransferase p300 in LECs in Ras-mediated transcriptional regulation of Vegfr3. Ras activates Ets proteins via MAPK-induced phosphorylation. Ets knockdown, similar to Ras knockdown, resulted in a decrease in both Vegfr3 transcript levels and acetylated histone H3 on the Vegfr3 gene. Vegfr3 knockdown results in altered LEC phenotypes, such as aberrant cell proliferation and network formation, and Ets knockdown led to milder but similar phenotypic changes. We identified evolutionarily conserved, non-coding regulatory elements within the Vegfr3 gene that harbor Ets-binding motifs and have enhancer activities in LECs. Chromatin immunoprecipitation (ChIP) assays revealed that acetylated histone H3 on the regulatory elements of the Vegfr3 gene was decreased following Ras and Ets knockdown, and that activated Ets proteins, together with p300, were associated with these regulatory elements, consistent with a reduction in Vegfr3 gene expression in p300-knockdown LECs. Our findings demonstrate a link between Ras signaling and Ets- and p300-mediated transcriptional regulation of Vegfr3, and provide a potential mechanism by which VEGFR-3 expression levels may be modulated during lymphangiogenesis.

Phospholipase Cgamma2 is necessary for separation of blood and lymphatic vasculature in mice.

The lymphatic vasculature originates from the blood vasculature through a mechanism relying on Prox1 expression and VEGFC signalling, and is separated and kept separate from the blood vasculature in a Syk- and SLP76-dependent manner. However, the mechanism by which lymphatic vessels are separated from blood vessels is not known. To gain an understanding of the vascular partitioning, we searched for the affected gene in a spontaneous mouse mutant exhibiting blood-filled lymphatic vessels, and identified a null mutation of the Plcg2 gene, which encodes phospholipase Cgamma2 (PLCgamma2), by positional candidate cloning. The blood-lymph shunt observed in PLCgamma2-null mice was due to aberrant separation of blood and lymphatic vessels. A similar phenotype was observed in lethally irradiated wild-type mice reconstituted with PLCgamma2-null bone marrow cells. These findings indicate that PLCgamma2 plays an essential role in initiating and maintaining the separation of the blood and lymphatic vasculature.