Expression of a dominant negative FGF receptor in developing GNRH1 neurons disrupts axon outgrowth and targeting to the median eminence

During development, neurons that synthesize and release gonadotropin-releasing hormone (GNRH1) extend their axons to the median eminence (ME) to establish neurosecretory contacts necessary for hormone secretion. Signals that coordinate this process are not known, but could involve the activation of fibroblast growth factor receptors (FGFRs) expressed on developing GNRH1 neurons. Using both whole-animal and cell culture approaches, this study examines the direct role of FGFR signaling in the extension and guidance of GNRH1 axons to the ME. In vivo retrograde labeling with fluorogold (FG) first showed a significant reduction in the projections of GNRH1 axons to the circumventricular organs (including the ME) in transgenic mice expressing a dominant negative FGF receptor (dnFGFR) in GNRH1 neurons. Using a primary GNRH1 neuronal culture system, we examined if compromised axon extension and directional growth led to the reduced axon targeting efficiency seen in vivo. Primary cultures of GNRH1 neurons were established from Embryonic Day 15.5 embryos, an age when GNRH1 neurons are actively targeting the ME. Cultured GNRH1 neurons expressing dnFGFR (dnFGFR neurons) exhibited attenuated activation of signaling pathways and reduced neurite outgrowth in response to FGF2. Further, dnFGFR neurons failed to preferentially target neurites toward cocultured ME explant and FGF2-coated beads, suggesting a defect in axon pathfinding. Together, these findings describe a direct role of FGFR signaling in the elongation and guidance of GNRH1 axons to the ME.     

Characterization of sequences and mechanisms through which ISE/ISS-3 regulates FGFR2 splicing

Alternative splicing of fibroblast growth factor receptor-2 (FGFR2) mutually exclusive exons IIIb and IIIc results in highly cell-type-specific expression of functionally distinct receptors, FGFR2-IIIb and FGFR2-IIIc. We previously identified an RNA cis-element, ISE/ISS-3, that enhanced exon IIIb splicing and silenced exon IIIc splicing. Here, we have performed comprehensive mutational analysis to define critical sequence motifs within this element that independently either enhance splicing of upstream exons or repress splicing of downstream exons. Such analysis included use of a novel fluorescence-based splicing reporter assay that allowed quantitative determination of relative functional activity of ISE/ISS-3 mutants using flow cytometric analysis of live cells. We determined that specific sequences within this element that mediate splicing enhancement also mediate splicing repression, depending on their position relative to a regulated exon. Thus, factors that bind the element are likely to be coordinately involved in mediating both aspects of splicing regulation. Exon IIIc silencing is dependent upon a suboptimal branchpoint sequence containing a guanine branchpoint nucleotide. Previous studies of exon IIIc splicing in HeLa nuclear extracts demonstrated that this guanine branchsite primarily impaired the second step of splicing suggesting that ISE/ISS-3 may block exon IIIc inclusion at this step. However, results presented here that include use of newly developed in vitro splicing assays of FGFR2 using extracts from a cell line expressing FGFR2-IIIb strongly suggest that cell-type-specific silencing of exon IIIc occurs at or prior to the first step of splicing.     

Activating Somatic FGFR2 Mutations in Breast Cancer

It is known that FGFR2 gene variations confer a risk for breast cancer. FGFR2 and FGF10, the main ligand of FGFR2, are both overexpressed in 5–10% of breast tumors. In our study, we sequenced the most important coding regions of FGFR2 in somatic tumor tissue of 140 sporadic breast cancer patients and performed MLPA analysis to detect copy number variations in FGFR2 and FGF10. We identified one somatic heterozygous missense mutation, p.K660N (c.1980G>C), within the tyrosine kinase domain of FGFR2 in tumor tissue of a sporadic breast cancer patient, which is likely mediated by the FGFR2-IIIb isoform. The presence of wild type and mutated alleles in equal quantities suggests that the mutation has driven clonal amplification of mutant cells. We have analyzed the tyrosine kinase activity of p.K660N and another recently described somatic breast cancer mutation in FGFR2, p.R203C, after expression in HEK293 cells and demonstrated that the intrinsic tyrosine kinase activity of both mutant proteins is strongly increased resulting in elevated phosphorylation and activity of downstream effectors. To our knowledge, this is the first report of functional analysis of somatic breast cancer mutations in FGFR2 providing evidence for the activating nature of FGFR2-mediated signalling in the pathogenesis of breast cancer.     

FGFR1-IIIb is a putative marker of pancreatic progenitor cells

The pancreas develops from buds that derive from the endodermal epithelium of the digestive tract. The progenitor cells that will give rise to the mature pancreatic cells reside within this epithelium. However, their exact identity remains unknown. In the present study, we searched for genes expressed by pancreatic progenitor cells. We focused our search on receptor tyrosine kinases. We found that fibroblast growth factor-IIIb (FGFR1-IIIb) expression is high in pancreatic epithelium enriched in progenitor cells. We next investigated FGFR1-IIIb expression throughout pancreatic development. At early stages of pancreas development, FGFR1-IIIb is expressed by pancreatic epithelial cells that resemble undifferentiated cells, while at later stages of development, FGFR1-IIIb expression decreases, concomitant with the expected decrease in the number of progenitor cells.     

Fibroblast growth factor receptor 1-IIIb is dispensable for skin morphogenesis and wound healing

Alternative splicing in the extracellular domain is a characteristic feature of members of the fibroblast growth factor receptor (FGFR) family. This splicing event generates receptor variants, which differ in their ligand binding specificities. A poorly characterized splice variant is FGFR1-IIIb, recently found to be a functional FGF receptor predominantly expressed in the skin. Here we show that FGFR1-IIIb is expressed in normal and wounded mouse skin. Reduced expression of this type of receptor was found in wounds of healing-impaired genetically diabetic mice, suggesting that downregulation of FGFR1-IIIb is associated with wound healing defects. To address this possibility, we deleted the IIIb exon of FGFR1 in mice. The lack of FGFR-IIIb did not alter the expression of either FGFR1-IIIc, other FGF receptor genes or of FGFR1-IIIb ligands in normal and wounded skin. Histological analysis of the skin of FGFR1-IIIb knockout animals did not reveal any obvious abnormalities. Furthermore, full-thickness excisional skin wounds in these mice healed normally and no defects could be observed at the macroscopic or histological level. Finally, several genes that encode key players in wound repair were normally expressed in these animals. These data demonstrate that FGFR1-IIIb is dispensable for skin development and wound repair.     

FGF signals specifically regulate the structure of hair shaft medulla via IGF-binding protein 5

Reciprocal interactions between the dermal papilla and the hair matrix control proliferation and differentiation in the mature hair follicle. Analysis of expression suggests an important role for FGF7 and FGF10, as well as their cognate receptor FGFR2-IIIb, in these processes. Transgenic mice that express a soluble dominant-negative version of this receptor in differentiating hair keratinocytes were generated to interfere with endogenous FGF signalling. Transgenic mice develop abnormally thin but otherwise normal hairs, characterised by single columns of medulla cells in all hair types. All structural defects and the accompanying changes of global gene expression patterns are restricted to the hair medulla. Forced transgenic expression of IGF-binding protein 5, whose expression level is elevated upon suppression of FGFR2-IIIb-mediated signalling largely phenocopies the defect of dnFgfr2-IIIb-expressing hairs. Thus, the results identify Igfbp5-mediated FGFR2-IIIb signals as a key regulator of the genetic program that controls the structure of the hair shaft medulla.     

Production of Transgenic-clone Pigs by the Combination of ICSI-mediated Gene Transfer with Somatic Cell Nuclear Transfer

The objective of this study was to examine whether the ICSI-mediated gene transfer method using in vitro matured oocytes and frozen sperm head could actually produce transgenic pigs. We also aimed at examining whether transgenic pigs can be cloned from somatic cells of a transgenic pig generated by the ICSI-mediated method. A bicistronic gene constituted of the human albumin (hALB) and enhanced green fluorescent protein (EGFP) genes was introduced into pig oocytes by the ICSI-mediated method. Transfer of 702 embryos produced by the ICSI-mediated method into five gilts resulted in 4 pregnancies. When three of the recipients, which had received total 312 of the embryos were autopsied, 32 including 1 transgenic fetuses were obtained. One of the recipients gave birth to three live piglets including one transgenic pig, showing a strong green fluorescence in the eyeballs, oral mucous membrane and subcutaneous tissues. Fluorescent microscopy revealed uniform GFP expression in all cell lines established from kidney, lung and muscle of the founder transgenic pig obtained. Nuclear transfer of these cells resulted in stable in vitro development of cloned embryos into the blastocyst stage, ranging from 12.9 to 19.8%. When 767 of the nuclear transfer embryos were transferred to 5 recipients, all became pregnant and gave birth to a total of six live transgenic-clones. The transgene copy number and integrity in the founder pig were maintained in the primary culture cells established from the founder as well as in the clones produced from these cells. Our study demonstrates that the ICSI-mediated gene transfer is an efficient and practical method to produce transgenic pigs, using frozen sperm heads and in vitro matured oocytes. It was also shown that combination of ICSI-mediated transgenesis and nuclear transfer is a feasible technology of great potential in transgenic pig production.     

Fibroblast Growth Factor 10-Fibroblast Growth Factor Receptor 2b Mediated Signaling Is Not Required for Adult Glandular Stomach Homeostasis

The signaling pathways that are essential for gastric organogenesis have been studied in some detail; however, those that regulate the maintenance of the gastric epithelium during adult homeostasis remain unclear. In this study, we investigated the role of Fibroblast growth factor 10 (FGF10) and its main receptor, Fibroblast growth factor receptor 2b (FGFR2b), in adult glandular stomach homeostasis. We first showed that mouse adult glandular stomach expressed Fgf10, its receptors, Fgfr1b and Fgfr2b, and most of the other FGFR2b ligands (Fgf1, Fgf7, Fgf22) except for Fgf3 and Fgf20. Fgf10 expression was mesenchymal whereas FGFR1 and FGFR2 expression were mostly epithelial. Studying double transgenic mice that allow inducible overexpression of Fgf10 in adult mice, we showed that Fgf10 overexpression in normal adult glandular stomach increased epithelial proliferation, drove mucous neck cell differentiation, and reduced parietal and chief cell differentiation. Although a similar phenotype can be associated with the development of metaplasia, we found that Fgf10 overexpression for a short duration does not cause metaplasia. Finally, investigating double transgenic mice that allow the expression of a soluble form of Fgfr2b, FGF10''s main receptor, which acts as a dominant negative, we found no significant changes in gastric epithelial proliferation or differentiation in the mutants. Our work provides evidence, for the first time, that the FGF10-FGFR2b signaling pathway is not required for epithelial proliferation and differentiation during adult glandular stomach homeostasis.     

Cyclooxygenase-2 in newborn hyperoxic lung injury

Supraphysiological O₂ concentrations, mechanical ventilation, and inflammation significantly contribute to the development of bronchopulmonary dysplasia (BPD).Exposure of newborn mice to hyperoxia causes inflammation and impaired alveolarization similar to that seen in infants with BPD.Previously, we demonstrated that pulmonary cyclooxygenase-2 (COX-2) protein expression is increased in hyperoxia-exposed newborn mice.The present studies were designed to define the role of COX-2 in newborn hyperoxic lung injury.We tested the hypothesis that attenuation of COX-2 activity would reduce hyperoxia-induced inflammation and improve alveolarization.Newborn C3H/HeN micewere injected daily with vehicle, aspirin (nonselective COX-2 inhibitor), or celecoxib (selective COX-2 inhibitor) for the first 7 days of life.Additional studies utilized wild-type (C57Bl/6, COX-2^+/+), heterozygous (COX-2^+/-), and homozygous (COX-2^-/-) transgenic mice.Micewere exposed to room air (21% O₂) or hyperoxia (85% O₂) for 14 days.Aspirin-injected and COX-2^-/- pups had reduced levels of monocyte chemoattractant protein (MCP-1) in bronchoalveolar lavage fluid (BAL).Both aspirin and celecoxib treatment reduced macrophage numbers in the alveolar walls and air spaces.Aspirin and celecoxib treatment attenuated hyperoxia-induced COX activity, including altered levels of prostaglandin (PG)D₂ metabolites.Decreased COX activity, however, did not prevent hyperoxia-induced lung developmental deficits.Our data suggest thatincreased COX-2 activity may contribute to proinflammatory responses, including macrophage chemotaxis, during exposure to hyperoxia.Modulation of COX-2 activity may be a useful therapeutic target to limit hyperoxia-induced inflammation in preterm infants at risk of developing BPD.     

Lean and obese pig breeds exhibit differences in prenatal gene expression profiles of muscle development

Muscle development in domesticated animals is important for meat production. Furthermore, intramuscular fat content is an important trait of meat intended for consumption. Here, we examined differences in the expression of factors related to myogenesis, adipogenesis and skeletal muscle growth during fetal muscle development of lean (Yorkshire) and obese (Chenghua) pig breeds. At prenatal days 50 (d50) and 90 (d90), muscles and sera were collected from pig fetuses. Histology revealed larger diameters and numbers of myofibers in Chenghua pig fetuses than those in Yorkshire pig fetuses at d50 and d90. Yorkshire fetuses had higher serum concentrations of myostatin (d90), a negative regulator for muscle development, and higher mRNA expression of the growth hormone receptor Ghr (d90), myogenic MyoG (d90) and adipogenic LPL (d50). By contrast, Chenghua fetuses exhibited higher serum concentration of growth hormone (d90), and higher mRNA expression of myogenic MyoD (d90) as well as adipogenic PPARG and FABP4 (d50). Our results revealed distinct expression patterns in the two pig breeds at each developmental stage before birth. Compared with Chenghua pigs, development and maturation of fetal skeletal muscles may occur earlier in Yorkshire pigs, but the negative regulatory effects of myostatin may suppress muscle development at the later stage.     

The roles of FGF signaling in germ cell migration in the mouse

Fibroblast growth factor (FGF) signaling is thought to play a role in germ cell behavior. FGF2 has been reported to be a mitogen for primordial germ cells in vitro, whilst combinations of FGF2, steel factor and LIF cause cultured germ cells to transform into permanent lines of pluripotent cells resembling ES cells. However, the actual function of FGF signaling on the migrating germ cells in vivo is unknown. We show, by RT-PCR analysis of cDNA from purified E10.5 germ cells, that germ cells express two FGF receptors: Fgfr1-IIIc and Fgfr2-IIIb. Second, we show that FGF-mediated activation of the MAP kinase pathway occurs in germ cells during their migration, and thus they are potentially direct targets of FGF signaling. Third, we use cultured embryo slices in simple gain-of-function experiments, using FGF ligands, to show that FGF2, a ligand for FGFR1-IIIc, affects motility, whereas FGF7, a ligand for FGFR2-IIIb, affects germ cell numbers. Loss of function, using a specific inhibitor of FGF signaling, causes increased apoptosis and inhibition of cell shape change in the migrating germ cells. Lastly, we confirm in vivo the effects seen in slice cultures in vitro, by examining germ cell positions and numbers in embryos carrying a loss-of-function allele of FGFR2-IIIb. In FGFR2-IIIb(-/-) embryos, germ cell migration is unaffected, but the numbers of germ cells are significantly reduced. These data show that a major role of FGF signaling through FGFR2-IIIb is to control germ cell numbers. The data do not discriminate between direct and indirect effects of FGF signaling on germ cells, and both may be involved.     

Conditional overexpression of TGFβ1 promotes pulmonary inflammation,apoptosis and mortality via TGFβR2 in the developing mouse lung

Background

Earlier studies have reported that transforming growth factor beta 1(TGFβ1) is a critical mediator of hyperoxia-induced acute lung injury (HALI) in developing lungs, leading to impaired alveolarization and a pulmonary phenotype of bronchopulmonary dysplasia (BPD). However, the mechanisms responsible for the TGFβ1-induced inflammatory signals that lead to cell death and abnormal alveolarization are poorly understood. We hypothesized that TGFβ1 signaling via TGFβR2 is necessary for the pathogenesis of the BPD pulmonary phenotype resulting from HALI.

Methods

We utilized lung epithelial cell-specific TGFβ1 overexpressing transgenic and TGFβR2 null mutant mice to evaluate the effects on neonatal mortality as well as pulmonary inflammation and apoptosis in developing lungs. Lung morphometry was performed to determine the impaired alveolarization and multicolor flow cytometry studies were performed to detect inflammatory macrophages and monocytes in lungs. Apoptotic cell death was measured with TUNEL assay, immunohistochemistry and western blotting and protein expression of angiogenic mediators were also analyzed.

Results

Our data reveals that increased TGFβ1 expression in newborn mice lungs leads to increased mortality, macrophage and immature monocyte infiltration, apoptotic cell death specifically in Type II alveolar epithelial cells (AECs), impaired alveolarization, and dysregulated angiogenic molecular markers.

Conclusions

Our study has demonstrated the potential role of inhibition of TGFβ1 signaling via TGFβR2 for improved survival, reduced inflammation and apoptosis that may provide insights for the development of potential therapeutic strategies targeted against HALI and BPD.     

Loss of FGF receptor 1 signaling reduces skeletal muscle mass and disrupts myofiber organization in the developing limb

The identities of extracellular growth factors that regulate skeletal muscle development in vivo are largely unknown. We asked if FGFs, which act as repressors of myogenesis in culture, play a similar role in vivo by ectopically expressing in the developing limb a truncated FGF receptor 1 (dnFGFR1) that acts as a dominant negative mutant. Hind limbs and the adjacent somites of Hamburger and Hamilton (HH) stage 17 chickens were infected with a replication-competent RCAS virus encoding dnFGFR1. By ED5, the virus had spread extensively within the limb and the adjacent somites with little rostral or caudal expansion of the infection along the axial midline. Viral infection and mutant receptor expression were coincident as revealed by the distribution of a viral coat protein and an HA epitope tag present on the carboxy terminus of dnFGFR1. Within 48 h following injection of dnFGFR1, we could detect no obvious changes in skeletal muscle precursor cell migration into the hind limb as compared to control limbs infected with an empty RCAN virus. However, by 3 days following infection of RCAS-dnFGFR1 virus, the level of skeletal muscle-specific myosin heavy chain was decreased and the expression pattern altered, suggesting disruption of skeletal muscle development. Two striking muscular phenotypes were observed in dnFGFR1-expressing limbs, including an average loss of 30% in skeletal muscle wet weight and a 50% decrease in myofiber density. At all ages examined the loss of skeletal muscle mass was accompanied by a loss of myoblasts and an unexpected concomitant loss of fibroblasts. Consistent with these observations, explants of infected cells revealed a reduction in the number of myonuclei in myotubes. Although the myofiber density per unit area was decreased over 50% compared to controls there were no detectable effects on myofiber diameter. The loss in myofiber density was, however, accompanied by an increase in the space surrounding individual myofibers and a generalized loss of myofiber integrity. It is noteworthy that long-bone development was unaffected by RCAS-dnFGFR1 infection, suggesting that FGFR2 and FGFR3 signaling was not disrupted. Our data provide conclusive evidence that FGFR1 signaling is necessary to maintain myoblast number and plays a role in myofiber organization.