FGF-2 mRNA Research Articles

230 EXPRESSION OF mRNA ENCODING FGF10 AND COGNATE RECEPTORS (FGFR1B AND FGFR2B) AROUND FOLLICLE DEVIATION IN NELORE HEIFERS

A member of the FGF7 subfamily, FGF10 acts via FGFR2B and FGFR1B. In bovine antral follicles, FGF-10 was detected in oocytes and theca cells (TC). Levels of mRNA were negatively correlated with intrafollicular concentrations of estradiol, and FGF10 inhibited estradiol production from granulosa cells (GC). In Nellore (Bos indicus), morphological divergence occurs on average 2.5 days after ovulation, when dominant follicle diameter is around 6.0 mm. To gain insight into the involvement of the FGF10 system in the control of follicle selection, we assessed mRNA expression of FGF10 in TC and of FGFR1B and FGFR2B in GC from dominant and subordinate follicles around deviation in Nellore heifers. Thirteen Nellore heifers were hormonally synchronized, and ovulation was detected by ultrasound monitoring every 12 h. Heifers were slaughtered 2 (n =4), 2.5 (n = 5), and 3 (n = 4) days after ovulation. Granulosa cells and TC were separated from the 2 largest follicles and submitted to total RNA extraction. mRNA abundance of CYP19 (aromatase), FGF10, FGFR1B, and FGFR2B was measured by real-time RT-PCR and normalized by the expression of cyclophilin A (CYCA) and GAPDH, for TC and GC, respectively. Dominant and subordinate follicles were considered those expressing the greatest and second-greatest abundance of CYP19 mRNA in GC within each heifer. Effects of follicle status and day on CYP19, FGF10, FGFR2B, and FGFR1B mRNA abundance were tested by ANOVA. On Day 2, FGFR2B mRNA abundance was greater in GC of subordinate follicles compared with dominant follicles (P = 0.006), and that of FGF10 in TC tended to exhibit the same pattern (P = 0.06). Follicle diameter was not different between dominant and subordinate follicles on Day 2 (5.5 ± 0 v. 5.12 ± 0.3 cm). On Day 2.5, FGF10 expression was greater in TC from subordinate follicles (P = 0.01), and FGFR2B expression in GC was no longer different between dominant and subordinate follicles. Follicle diameter was greater in dominant follicles on Day 2.5 (6.7 ± 0.2 v. 5.8 ± 0.3 cm; P = 0.04). On Day 3, no differences were observed between dominant and subordinate follicles for any of the genes assessed. mRNA expression of FGFR1B in GC did not change with follicle status or day. In conclusion, expression of FGF10 and FGFR2B was decreased in dominant follicles around morphological divergence, suggesting their involvement in the mechanisms controlling dominant follicle selection. As FGF10 inhibits estradiol production of GC, we propose that FGF10 and FGFR2B are suppressed in the dominant follicle to allow acquisition of full steroidogenic capacity. This research was supported by FAPESP.

Assessment of the Impact of Enforced Expression of Platelet Derived Growth Factor Beta in Human Mesenchymal Stem Cells and Contribution to Blood Flow Restoration.

Abstract 3568Poster Board III-505Both mesenchymal stem cells (MSC) and platelet derived growth factor beta (PDGFB) have been shown to promote angiogenesis in vivo and are therefore interesting therapeutic agents for the restoration of blood flow to ischemic tissues encountered in peripheral artery disease and critical limb ischemia. Careful evaluation of the mechanisms responsible for neovessel formation in stem cell-based therapeutic angiogenesis is critical for developing efficient treatment strategies. Previous studies have shown that MSCs correspond to pericytes, which are lost in PDGFB-null mice, suggesting that MSCs strongly rely on PDGFB signaling in vivo. Our major aim was to establish a highly sensitive method to assess whether overexpression of PDGFB in MSCs could modulate expression of pro-angiogenic cytokines and improve the restoration of bloodflow in an established murine xenograft model of hind limb ischemia. Human bone marrow derived MSCs (passage 2-5) were transduced with either a lentiviral vector containing the coding sequence of PDGFB or the same vector without PDGFB (control). As expected, overexpression of PDGFB strongly enhanced the proliferation of MSCs, reducing the doubling time from 51 to 20 hours (n=4, p<0.05). This effect correlated with a shift of 15% of cells from the G0/G1 to the S/G2/M phase, as compared to controls, when measured by flow cytometry of propidium iodide stained cells. Next, we induced a unilateral hind limb ischemia in NODSCIDbeta2null mice by excision of the femoral artery and performed intravenous transplantation of 2.5×10e5 MSCs (control MSC vs. PDGFB-MSC) the day following surgery. Recovery of blood flow to the affected limb was assessed at days 0 ((before transplantation), n=11), 4 (n=11), 7 (n=9), 10 (n=6), and 14 (n=6) by laser doppler imaging and values were normalized to the non-ischemic contralateral leg. Treated animals of both groups (control and PDGFB overexpressing MSC) exhibited a steadily progressive recovery in blood flow in the region proximal to the primary ligation site, reaching near normal levels by day 10. Surprisingly, no significant difference was seen in the blood flow recovery in animals receiving the PDGF over expressing MSCs. Donor MSCs (control and PDGFB) homed to the site of injury, as assessed by immunofluorescent staining of the ischemic muscle in the regions proximal and distal to the primary ligation site at days 4 and 7. However, the rapid recovery in blood flow did not appear to be attributed to a direct differentiation or integration of MSC into the vasculature, but rather through the expression of paracrine angiogenic factors. Interestingly, overexpression of PDGF reduced FGF2 mRNA expression levels in MSC (n=4, p=0.056), suggesting that high levels of PDGFB may indeed lead to a reduction of other key angiogenic factors, explaining the mild effect of PDGFB in vivo. Our results show that, although overexpression of PDGFB in bone marrow-derived MSCs enhanced the proliferation of MSC in vitro, it did not impact the speed or magnitude of blood flow recovery to the ischemic hind limb as compared to native MSCs, likely due to down modulation of other key angiogenic signals including FGF2. Disclosures:No relevant conflicts of interest to declare.

TGF-β1 RNA Interference in Mouse Primary Dura Cell Culture: Downstream Effects on TGF Receptors, FGF-2, and FGF-R1 mRNA Levels

Transforming growth factor (TGF)-beta1 and fibroblast growth factor (FGF)-2 have both been shown to have significant roles in the regulation of murine calvarial suture fusion. Methods to decrease gene expression of these cytokines and their respective receptors have been established, but because of side effects, clinical applications are limited. In this study, the authors examined the effect of TGF-beta1-specific small interfering RNA (siRNA) on the messenger RNA (mRNA) expression of TGF-beta1, its TGF-betaR1 and TGF-betaR2 receptors, and FGF-2 and its R1 receptor in murine dura cells. A primary dura cell line was established from CD-1 mice. Transfection efficiency using Lipofectamine was determined using BLOCKiT. Dura cells were transfected with serial concentrations of TGF-beta1 siRNA to determine the optimal dose. In subsequent experiments, cells were transfected with 16 nM TGF-beta1 siRNA and harvested on posttransfection days 4, 7, 10, and 14 for RNA isolation and quantitative polymerase chain reaction. Optimal inhibition of TGF-beta1 mRNA expression was achieved at 16 nM siRNA. On posttransfection day 4, TGF-beta1 mRNA levels were significantly decreased but returned to baseline by day 14. TGF-betaR1 mRNA expression remained unaffected by transfection throughout the time course. However, TGF-betaR2, FGF-2, and FGF-R1 demonstrated significant inhibition of mRNA expression on posttransfection day 4. These results indicate that TGF-beta1 siRNA has the potential to alter the murine dura cytokines responsible for suture fusion in vitro. Manipulating underlying cranial suture biology with siRNA technology may ultimately allow control over suture fusion. This intervention may ultimately function as an effective adjunct to surgical intervention for craniosynostosis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Inhibits Fibroblast Growth Factor 10-Induced Prostatic Bud Formation in Mouse Urogenital Sinus

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) dorsalizes the pattern of prostatic buds developing from the urogenital sinus (UGS) of male fetal mice, causing some buds to form in inappropriate positions while blocking formation of others. This teratogenic TCDD action significantly reduces prostate main duct number and causes ventral prostate agenesis in exposed males. The purpose of this study was to determine whether inhibition of fibroblast growth factor 10 (FGF10) signaling is mechanistically linked to mouse prostatic budding impairment by TCDD. In utero TCDD exposure induced aryl hydrocarbon receptor-responsive cytochrome P450 1b1 messenger RNA (mRNA) in ventral UGS regions where Fgf10 and fibroblast growth factor receptor 2 (Fgfr2) mRNA were expressed and where budding was most severely inhibited by TCDD. However, TCDD exposure did not reduce Fgf10 or Fgfr2 mRNA abundance in the UGS or alter their distribution. Addition of FGF10 protein to UGS organ culture media increased the abundance of UGS basal epithelial cells immunopositive for phosphorylated extracellular signal-regulated kinase (ERK). FGF10 also increased the number of 5-bromo-2'-deoxyuridine (BrdU)-labeled UGS epithelial cells and increased the number of prostatic buds formed per UGS. Addition of TCDD to UGS organ culture media did not alter FGF10-induced ERK activation in UGS basal epithelium but prevented FGF10-induced BrdU incorporation and blocked FGF10-induced prostatic bud formation. These results identify basal urogenital sinus epithelium cells as the key site of FGF10 action during fetal prostate development and suggest that TCDD likely acts downstream of FGFR2 and ERK to restrict UGS epithelial cell proliferation and prevent prostatic bud formation.

Exercise in Patients with Intermittent Claudication Elicits Signs of Inflammation and Angiogenesis

Previous studies have demonstrated elevation of systemic levels of inflammatory cytokines after treadmill exercise in patients with intermittent claudication (IC), but it is unknown if growth factor expression also is stimulated. The aim of this study was to assess whether physical exercise-induced ischemia elicits an inflammatory response and increase in local and systemic vascular growth factor expression in patients with IC. Nineteen patients with IC had plasma concentrations of inflammatory markers (IL-6, TNF-alpha, hs-CRP) and vascular growth factors (VEGF and FGF-2) measured before and at four time points after a treadmill exercise test. In 10 patients a gastrocnemius muscle biopsy was obtained to measure VEGF and FGF-2 mRNA. Plasma concentrations of vWF were also measured. Five patients who underwent the treadmill test without experiencing calf pain were enrolled as controls. Plasma concentrations of IL-6 increased after exercise (p=0.004), while TNF-alpha and hs-CRP were unchanged (p=0.191 and p=0.709, respectively). Plasma concentrations of VEGF were similar (p=0.151) at the different time points after exercise but FGF-2 levels decreased (p=0.013). In biopsies after treadmill testing VEGF-A mRNA was increased (p=0.043), but no change was observed for FGF-2 (p=0.456). Exercise in IC triggers an inflammatory response as exemplified by elevated concentrations of IL-6. After exercise-induced pain, VEGF mRNA in calf muscle is increased. Therefore, it is plausible that angiogenesis is stimulated by exercise in IC.

Differences in the Nemosis Response of Normal and Cancer-Associated Fibroblasts from Patients with Oral Squamous Cell Carcinoma

BackgroundTumor-stroma reaction is associated with activation of fibroblasts. Nemosis is a novel type of fibroblast activation. It leads to an increased production of growth factors and proinflammatory and proteolytic proteins, while at the same time cytoskeletal proteins are degraded. Here we used paired normal skin fibroblasts and cancer-associated fibroblasts (CAF) and primary and recurrent oral squamous cell carcinoma (SCC) cells to study the nemosis response.Principal FindingsFibroblast nemosis was analyzed by protein and gene expression and the paracrine regulation with colony formation assay. One of the normal fibroblast strains, FB-43, upregulated COX-2 in nemosis, but FB-74 cells did not. In contrast, CAF-74 spheroids expressed COX-2 but CAF-43 cells did not. Alpha-SMA protein was expressed in both CAF strains and in FB-74 cells, but not in FB-43 fibroblasts. Its mRNA levels were downregulated in nemosis, but the CAFs started to regain the expression. FSP1 mRNA was downregulated in normal fibroblasts and CAF-74 cells, but not in CAF-43 fibroblasts. Serine protease FAP was upregulated in all fibroblasts, more so in nemotic CAFs. VEGF, HGF/SF and FGF7 mRNA levels were upregulated to variable degree in nemosis. CAFs increased the colony formation of primary tumor cell lines UT-SCC-43A and UT-SCC-74A, but normal fibroblasts inhibited the anchorage-independent growth of recurrent UT-SCC-43B and UT-SCC-74B cells.ConclusionsNemosis response, as observed by COX-2 and growth factor induction, and expression of CAF markers α-SMA, FSP1 and FAP, varies between fibroblast populations. The expression of CAF markers differs between normal fibroblasts and CAFs in nemosis. These results emphasize the heterogeneity of fibroblasts and the evolving tumor-promoting properties of CAFs.

Metabolic programming of ovarian angiogenesis and folliculogenesis by maternal malnutrition during lactation

To evaluate whether maternal malnutrition during lactation programs ovarian folliculogenesis and the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and its receptors KDR, Flt-1, and FGFR. Experimental study. University-based research laboratory. Adult female rats from a urogenital research laboratory. Six rat dams randomly assigned to the following groups: control group (C), with free access to a standard laboratory diet containing 23% protein; and a protein-energy-restricted group (PER), with free access to an isoenergy and protein-restricted diet containing 8% protein. After weaning, the female pups had free access to the standard laboratory diet until 90 days of age, when they were sacrificed at the proestrum stage. Quantification of ovarian follicles, vessels, and expression of growth factors and their receptors. Maternal malnutrition during lactation caused a significant reduction in the number of primordial (C = 6.60 +/- 0.24, PER = 5.20 +/- 0.20), primary (C = 5.80 +/- 0.66, PER = 4.00 +/- 0.31), and Graafian follicles/section (C = 2.18 +/- 0.29, PER = 1.08 +/- 0.37), in KDR (C = 0.22 +/- 0.04, PER = 0.09 +/- 0.01), Flt-1 (C = 0.28 +/- 0.05, PER = 0.12 +/- 0.02), and FGFR mRNA expression (C = 0.34 +/- 0.05, PER = 0.13 +/- 0.05) and in the vessel density of follicles (C = 17.26 +/- 2.30, PER = 9.96 +/- 0.97). Maternal malnutrition during lactation programs the follicular development by a reduction of VEGF and FGF mRNA receptors expression, probably from a direct action on the follicular development or a reduction in vasculature resulting in a decreased delivery of folliculotrophic substances in PER animals.

Follicle Atresia Is Regulated by Fibroblast Growth Factor-18 in Cattle.

Understanding the paracrine events that regulate fertility in the cow is necessary not only because of the agricultural importance of this species, but also its potential use as a model for humans. Fibroblast growth factors (FGF) and their receptors are involved in the paracrine modulation of ovarian function. For example, FGF2, 7 and 10 inhibit estradiol secretion by granulosa cells in vitro. Another member of this family, FGF8, is an oocyte-derived protein that modulates glycolysis in cumulus cells. FGFs are grouped into subfamilies of closely-related proteins, and FGF18 is in the same subfamily as FGF8 and has also been detected in mouse oocytes. The existance of a subfamily of FGFs that are specifically derived from the oocyte are potentially interesting paracrine regulators of fertility. The objectives of the present study were to localize the expression of FGF18 in the bovine follicle, to gain insight into the control of FGF18 expression and to identify potential actions of FGF18 in the follicle. Bovine ovaries were obtained from an abattoir for localizing expression patterns by real-time PCR and immunohistochemistry. FGF18 mRNA was not detected in pooled oocytes by PCR, but was present in isolated theca and granulosa cells (GC), and protein was detected in the follicle wall by immunohistochemistry. Expression during follicle growth was measured by collecting the two largest follicles per cow at defined stages of the first follicle wave. The follicles were classified as dominant and subordinate according to estradiol levels in follicular fluid and aromatase mRNA abundance in GC. FGF18 mRNA abundance in both GC and theca cells was higher in subordinate follicles compared with dominant follicles (P<0.05). To determine regulation and function of FGF18, granulosa cells were isolated from 5-8 mm follicles and employed in an estrogenic, serum-free culture system. In vitro, FGF18 expression was down-regulated by IGF1, insulin, FGF2, FGF10 (P<0.05) in GC. In further experiments, the addition of graded doses of FGF18 (0, 1, 10, 100 ng/ml) to GC in culture inhibited estradiol secretion and abundance of mRNA encoding the estrogenic enzymes aromatase and 17β-hydroxysteroid dehydrogenase (17βHSD), and the FSH receptor. Progesterone secretion was also inhibited, which was associated with decreased abundance of mRNA encoding CYP11, StAR and 3βHSD. Assessment of apoptosis by flow cytometry showed that FGF18 significantly increased the proportion of apoptotic cells, and this was accompanied by decreased abundance of mRNA encoding antiapoptotic and proliferation genes (MIF, RFC4 and GADD45B). In summary, FGF18 is expressed in GC and theca cells but--in contrast to the subfamily prototype, FGF8--not in oocytes in cattle. As FGF18 expression is increased in atretic follicles, and as FGF18 protein inhibits estradiol secretion and induces apoptosis, we conclude that FGF18 plays a role in follicular atresia. Supported by NSERC Canada, CNPq and FAPESP, Brazil. (platform)

Aryl hydrocarbon receptor activation and overexpression upregulated fibroblast growth factor‐9 in human lung adenocarcinomas

We had previously reported that aryl hydrocarbon receptors (AhRs) are overexpressed in lung adenocarcinomas. Benzo[a]pyrene (BaP), an AhR agonist, increased FGF-9 expression in human lung adenocarcinoma cells. Similarly, several AhR agonists increased FGF-9 mRNA levels, and BaP-induced FGF-9 expression was prevented by cotreatment with AhR antagonist in human lung adenocarcinoma cells. Furthermore, AhR agonists increased transcriptional activity of FGF-9 promoter. Modulation of AhR expression via RNA interference or transient overexpression respectively reduced or increased both constitutive and BaP-induced FGF-9 expression in human lung cells. These results suggested that AhR activation and overexpression increased FGF-9 expression in lung cells. FGF-9 increased growth of lung fibroblasts but not that of lung adenocarcinoma cells. However, conditioned media collected from FGF-9-treated fibroblasts increased cell growth of lung adenocarcinoma cells. Furthermore, lung adenocarcinoma cells expressed FGF receptor 2 and cotreatment with anti-FGF receptor 2 prevented the interaction between fibroblasts and tumor cells. It is likely that FGF-9-stimulated fibroblasts secreted unknown factors, which activated FGF receptor 2 and subsequently promoted growth of lung adenocarcinoma cells. We further compared AhR and FGF-9 expression in 146 non-small cell lung cancer (NSCLC) cases by immunohistochemistry. FGF-9 expression was more common in adenocarcinomas than in squamous cell carcinomas. Furthermore, FGF-9 and AhR expression were well correlated in lung adenocarcinomas. These results suggest that AhR expression correlated positively with FGF-9 expression in lung adenocarcinomas, which might promote tumor growth by modulating communication between tumor cells and fibroblasts. Preventing AhR overexpression or disturbing FGF-9 function may reduce the development of lung adenocarcinomas. (c) 2009 UICC.

Evidence that androgen-independent stromal growth factor signals promote androgen-insensitive prostate cancer cell growth in vivo

Activation of tumor-stromal interactions is considered to play a critical role in the promotion of tumorigenesis. To discover new therapeutic targets for hormone-refractory prostate tumor growth under androgen ablation therapy, androgen-sensitive LNCaP cells and the derived sublines, E9 (androgen-low-sensitive), and AIDL (androgen-insensitive), were recombined with androgen-dependent embryonic rat urogenital sinus mesenchyme (UGM). Tumors of E9 + UGM and AIDL + UGM were approximately three times as large as those of LNCaP + UGM. Tumors grown in castrated hosts exhibited reduced growth as compared with those in intact hosts. However, in castrated hosts, E9 + UGM and AIDL + UGM tumors were still approximately twice as large as those of LNCaP + UGM. Cell proliferation in tumors of E9 + UGM and AIDL + UGM grown in castrated host, was significantly higher than that in tumors of LNCaP + UGM. In vitro, expression of fibroblast growth factor (FGF)-2 and IGF-I, but not FGF-7 mRNA, was significantly reduced in UGM under androgen starvation. In cell culture, E9 cells were responsive to FGF-2 and FGF-7 stimulation, while AIDL responded to FGF-7 and IGF-1. Expression of FGFR1 and FGFR2 was considerably higher in E9 than those in LNCaP, similarly expression of FGFR2 and IGF-IR were elevated in AIDL. These data suggest that activation of prostate cancer cell growth through growth factor receptor expression may result in the activity of otherwise androgen-independent stromal growth factor signals such as FGF-7 under conditions of androgen ablation.

Role of basic fibroblast growth factor (FGF-2) in diabetic nephropathy and mechanisms of its induction by hyperglycemia in human renal fibroblasts

Basic fibroblast growth factor (FGF-2) plays a role in renal fibrogenesis, although its potential implications for tubulointerstitial involvement in diabetic nephropathy are unknown. We evaluated the expression of FGF-2 in kidney biopsies from patients with diabetic nephropathy and studied the mechanisms of its induction in human renal fibroblasts under hyperglycemia. Tubulointerstitial expression of FGF-2 was significantly upregulated in diabetic nephropathy compared with control kidneys with a good correlation to the degree of the injury. Fibroblasts cultivated in high glucose displayed increased FGF-2 mRNA as well as protein synthesis and secretion compared with normal glucose. Proliferation rates under hyperglycemia were significantly higher and could be almost completely inhibited by addition of a neutralizing FGF-2 antibody. Alterations in proliferation were associated with changes in p27(kip1) expression. Hyperglycemia induced the expression of PKC-beta1 and PKC-beta2; however, only inhibition of PKC-beta1 but not PKC-beta2 led to a significant decrease of FGF-2 levels. Relevance of the culture findings and functional association was corroborated by colocalization of FGF-2 and PKC-beta in human diabetic kidneys in vivo. High glucose stimulated fibronectin synthesis and secretion, which could be substantially prevented by inhibition of PKC-beta1 and to a lesser extent by inhibiting the FGF-2. Expression of active phosphorylated form of p38 mitogen-activated protein kinase was upregulated under hyperglycemia; however, its inhibition had no effects on FGF-2 synthesis. Our results implicate a role of FGF-2 in high glucose-altered molecular signaling in pathogenesis of diabetic renal disease.

Influence of retinoic acid on TBX1 expression in myocardial cells induced by Shh and Fgf8

The aim of this study was to explore the regulatory mechanism of retinoic acid (RA) on the TBX1 gene expression in myocardial cells. Ventricular cardiocytes were isolated from neonatal rats and cultured, and then treated with different concentrations of retinoic acid. The expression of Shh and Fgf8 at mRNA and protein levels in neonatal rat myocardial cells were measured by using RT-PCR and Western blot technique, respectively. There was basal expression of Shh and Fgf8 in the control group. When treated with 3 × 10−7 mol/L RA, we observed that the expression of Shh mRNA and protein in neonatal rat myocardial cells were up-regulated by 1.51 (P < 0.05) and 1.10 times (P < 0.05), respectively. In comparison with the control group, under the concentration of 5 × 10−7 mol/L RA, they were up-regulated by 2.21 (P < 0.05) and 2.38 times (P < 0.05) individually. Meanwhile, we could detect that the expression of Fgf8 mRNA and protein were up-regulated by 2.50 times (P < 0.05) and 80% (P < 0.05) separately compared with the control group after stimulation of 3 × 10−7 mol/L RA, and they were up-regulated by 3.48 (P < 0.05) and 2.04 times (P < 0.05) individually after stimulation of 5 × 10−7 mol/L RA. The results indicated that RA could induce the expression of Shh and Fgf8 in neonatal rat myocardial cells. At the same time, it has shown that Shh and Fgf8 were involved in the regulation process of RA on TBX1 expression.

Hopf bifurcation in the presomitic mesoderm during the mouse segmentation

Vertebrae and ribs arise from embryonic tissues called somites. Somites arise sequentially from the unsegmented embryo tail, called presomitic mesoderm (PSM). The pace of somite formation is controlled by gene products such as hairy and enhancer of split 7 (Hes7) whose expression oscillates in the PSM. In addition to the cyclic genes, there is a gradient of fibroblast growth factor 8 ( Fgf8) mRNA from posterior to anterior PSM. Recent experiments have shown that in the absence of Fgf signaling, Hes7 oscillations in the anterior and posterior PSM are lost. On the other hand, Notch mutants reduce the amplitude of posterior Hes7 oscillations and abolish anterior Hes7 oscillations. To understand these phenotypes, we delineated and simulated a logical and a delay differential equation (DDE) model with similar network topology in wild-type and mutant situations. Both models reproduced most wild-type and mutant phenotypes suggesting that the chosen topology is robust to explain these phenotypes. Numerical continuation of the model showed that even in the wild-type situation, the system changed from sustained to damped, i.e. a Hopf bifurcation occurred, when the Fgf concentration decreased in the PSM. This numerical continuation analysis further indicated that the most sensitive parameters for the oscillations are the parameters of Hes7 followed by those of Lunatic fringe (Lfng) and Notch1. In the wild-type, the damping of Hes7 oscillations was not so strong so that cells reached the new somites before they lose Hes7 oscillations. By contrast, in the fibroblast growth factor receptor 1 (Fgfr1) conditional knock-out (cKO) mutant simulation, Notch signaling was not able to maintain sustained Hes7 oscillations. Our analysis suggests that Fgf signaling makes cells enter an oscillatory state of Hes7 expression. After moving to the anterior PSM, where Fgf signaling is missing, Notch signaling compensates the damping of Hes7 oscillations in the anterior PSM.

Perichondrial expression of Wdr5 regulates chondrocyte proliferation and differentiation

Wdr5 is developmentally expressed in osteoblasts and is required for osteoblast differentiation. Mice overexpressing Wdr5 under the control of the mouse α(1)I collagen promoter (Col I-Wdr5) display accelerated osteoblast differentiation as well as accelerated chondrocyte differentiation, suggesting that overexpression of Wdr5 in osteoblasts affects chondrocyte differentiation. To elucidate the molecular mechanism by which overexpression of Wdr5 in the perichondrium regulates chondrocyte differentiation, studies were undertaken using skeletal elements and cultured metatarsals isolated from wild-type and Col I-Wdr5 embryos. FGF18 mRNA levels were decreased in Col I-Wdr5 humeri. Furthermore, local delivery of FGF18 to the bone collar of ex vivo cultures of metatarsals attenuated the chondrocyte phenotype of the Col I-Wdr5 metatarsals. Impairing local FGF action in wild-type metatarsals resulted in a chondrocyte phenotype analogous to that of Col I-Wdr5 metatarsals implicating impaired FGF action as the cause of the phenotype observed. The expression of Twist-1, which regulates chondrocyte differentiation, was increased in Col I-Wdr5 humeri. Chromatin immunoprecipitation analyses demonstrated that Wdr5 is recruited to the Twist-1 promoter. These findings support a model in which overexpression of Wdr5 in the perichondrium promotes chondrocyte differentiation by modulating the expression of Twist-1 and FGF18.

Gene targeted ablation of high molecular weight fibroblast growth factor‐2

Fibroblast growth factor-2 (FGF2) is produced as high molecular weight isoforms (HMW) and a low molecular weight isoform (LMW) by means of alternative usage of translation start sites in a single Fgf2 mRNA. Although the physiological function of FGF2 and FGF2 LMW has been investigated in myocardial capillarogenesis during normal cardiac growth, the role of FGF2 HMW has not been determined. Here, we report the generation of FGF2 HMW-deficient mice in which FGF2 HMW isoforms are ablated by the Tag-and-Exchange gene targeting technique. These mice are normal and fertile with normal fecundity, and have a normal life span. Histological, immunohistochemical, and morphometric analyses indicate normal myocardial architecture, blood vessel, and cardiac capillary density in young adult FGF2 HMW-deficient mice. These mice along with the FGF2- and FGF2 LMW-deficient mice that we have generated previously will be very useful for elucidating the differential functions of FGF2 isoforms in pathophysiology of cardiovascular diseases.

140. THE DEVELOPMENT OF THE PHALLUS IN A MARSUPIAL

The phallus, the limbs and the tail are all considered appendages in the developing mammal. In mice, several key genes including FGF8, BMP4, SHH, DLX5 and DLX6 are known to control the precise pattering of the limb and phallus in the fetus (reviewed in Yamada et al. 2006). The signalling cascade in both appendages begins with SHH interacting with FGF8. In humans, disruptions to these gene pathways result in malformations of both limbs and phallus because these appendages share conserved elements in patterning and development (Yamada et al. 2003). However, this is a poorly researched area so additional models are needed to provide a greater perspective into mammalian embryonic patterning especially of the external genitalia. In marsupials, since most sexual differentiation occurs after birth, the developing phallus and limbs are accessible at stages that occur in utero in humans and other eutherian mammals. We have used the tammar as an alternative model to understand the differentiation of the phallus and limb. FGF8, SHH and megalin mRNA and protein are all expressedin the limb and phallus from the early embryo until post-natal stages. SHH and megalin were co-localised in the urethral epithelium of the tammar phallus. This is the first study to detect megalin in the developing mammalian phallus. SHH is a critical gene in patterning the appendages, and megalin is a transport protein that ferries steroids into the cell where they can bind to their cognate receptor. It has a high affinity for the potent androgen androstanediol, an androgen critical for virilisation of the tammar phallus (Leihy et al. 2004). Further examination is underway to confirm whether the expression of the genes patterning the phallus in the mouse are conserved in the marsupial and whether megalin plays any role in the patterning of the phallus, possibly mediated by SHH.

YY1 Over-Expression in Human Brain Gliomas and Meningiomas Correlates with TGF-β1, IGF-1 and FGF-2 mRNA Levels

In this study we examined by QRT-PCR the mRNA expression of TGF-β 1, IGF-1, EGF, FGF-2 and YY1 in human brain tumors. Our findings introduce YY1, for the first time, as a novel gene implicated in brain gliomatogenesis and meningioma establishment. We present a positive correlation between the autocrine expression of YY1 and TGF-β 1, IGF-1 and FGF-2, known to be involved in the progression of gliomas and meningiomas. We suggest that mRNA profiling of the above genes in the early stages of disease development could be useful for prognostic purposes, and these genes can be considered as potential targets for therapeutic approaches against brain tumors.

Polysaccharide fraction of Agaricus brasiliensis avoids tumor-induced IL-10 production and changes the microenvironment of subcutaneous Ehrlich adenocarcinoma

Subcutaneous Ehrlich tumor-bearing mice were treated with in situ inoculation of a β-glucan-rich extract of Agaricus brasiliensis (ATF), which reduced tumor growth. Histopathological analysis showed that the tumor masses of control mice (Ehr) presented giant tumor cells and many mitotic figures whereas the tumor tissue obtained from ATF-treated animals (Ehr-ATF) presented a lower frequency of both mitotic and giant cells, associated with a higher frequency of apoptotic cells than Ehr. Analysis of the lymphoproliferative activity of spleen cells showed that the treatment had a suppressive rather than a stimulatory effect. Spleen cells of the Ehr group produced higher in vitro levels of IL-10 than normal controls and this occurrence was partially avoided by treatment with ATF. Analysis of cytokine production by tumor-infiltrating cells (ELISpot) showed that ATF induced a higher number of IFN-γ-producing cells at 7 and 14 days as well as reduction of IL-10-secreting cells at the latter time. Confocal microscopy analysis showed higher intensity of labeling of CD4+ and Mac-3+ cells in ATF-treated mice. Analysis of in situ expression of angiogenic growth factors showed a slight decrease of FGF-2 mRNA in Ehr-ATF animals (7th day) but not of VEGF-A or TGF-β expression. This fraction could not directly lyse either lymphocytes or tumor cells and we speculate that antitumor effect of ATF could be due to induction of a selective migration of immunocompetent cells from the spleen to the tumor site and to the switch of cytokine production.

Effects of FGF-1 and FGFR1 on the Genesis of Spontaneous Breast Cancer in TA2 Mice

Objective:To compare the expression of FGF-1 and FGFR1 in normal breast tissues,precancerous breast tissues and breast cancer tissues of TA2 mice,and to discuss the effect of FGF-1 and FGFR1 on the genesis of spontaneous breast cancer in TA2 mice.Methods:Normal breast tissues were obtained from normal TA2 female mice(12 cases).Precancerous breast tissues and breast cancer tissues were obtained from TA2 female mice with spontaneous breast cancer(17 cases).Immunohistochemistry was used to detect the expression of FGF-1,PCNA,and cyclin D1 proteins.Real time-PCR was employed to observe the expression of FGF-1 and FGFR1 mRNA.Results:The expression of FGF-1 mRNA and protein was higher in breast cancer and precancerous lesions than in the normal breast tissues(P0.05).No significant difference was found in FGF-1 expression between breast cancer tissues and precancerous lesions(P0.05).The expression of FGFR1 mRNA was higher in breast cancer tissues and precancerous lesions than in the normal breast tissues(P0.05).A significant difference was found in FGFR1 mRNA expression between breast cancer tissues and precancerous lesions(P=0.046).The expression of PCNA and cyclin D1 was higher in breast cancer tissues and precancerous lesions than in the normal breast tissues(P0.05).The expression of cyclin D1 was higher in breast cancer tissues than in precancerous lesions(P=0.001).Conclusion:FGF-1 and FGFR1 may contribute to the genesis of spontaneous breast cancer in TA2 mice by regulating cell cycle and promoting cell proliferation.

106 ANGIOGENIC FACTOR mRNA EXPRESSION IN FETAL MEMBRANES IN EARLY PREGNANT SHEEP

Maternal and fetal placental development may be compromised by use of assisted reproductive techniques, including cloning, resulting in poor placental angiogenesis and subsequent high embryonic/fetal loss (Palmieri et al. 2007 Placenta 28, 577–584). Before changes in vascular development in placenta from compromised pregnancies can be understood, a detailed knowledge of regulation of angiogenesis in placental tissues from normal pregnancies is necessary. Therefore, this study determined the expression pattern of mRNA for several angiogenic factors and their receptors: vascular endothelial growth factor (VEGF) and receptor (R) 1 and 2; basic fibroblast growth factor (FGF2) and FGFRIIIc; angiopoietin (ANGPT) 1 and 2 and ANGPTR (Tie2); endothelial nitric oxide synthase (eNOS) and NO receptor GUCY1B3 in fetal membranes (FM; fetal placenta) collected on Days 16, 18, 20, 22, 24, 26, 28, and 30 after mating (n = 5 to 8/day). Fetal membranes were snap frozen for evaluation of gene expression using quantitative, real-time RT-PCR. VEGF mRNA was increased (P &lt; 0.05) 2-fold on Days 28 and 30 compared with Days 16, 18, and 20, whereas VEGFR1 mRNA increased (P &lt; 0.05) 25- to 50-fold on Days 28 and 30 compared with Day 16, and VEGFR2 mRNA was greatest (P &lt; 0.05) on Day 22 compared with Days 16, 18, 28, and 30. FGF2 mRNA was 4-fold greater (P &lt; 0.05) on Day 22 compared with Day 16; however, FGF2RIIIc was unchanged from Day 16 through 30. eNOS mRNA was greatest (P &lt; 0.05) on Days 22 and 24 compared with Days 16 and 18, but GUCY1B3 mRNA was greatest (P &lt; 0.05) on Day 18 compared with Days 20, 24, and 28. ANGPT1 mRNA increased (P &lt; 0.05) 40-fold by Days 28 and 30 compared with Days 16 and 18. ANGPT2 mRNA was undetectable on Day 16, and increased (P &lt; 0.05) 5-fold from Days 18 through 30. ANGPTR mRNA was greatest (P &lt; 0.05) on Days 22 and 24 compared with Days 16 and 18. This description of expression of factors potentially regulating early placental angiogenesis during normal pregnancy in sheep will provide the foundation for understanding the dramatic increases in capillary cell proliferation and capillary size we have previously observed (unpublished) and for determining whether placental vascular development is altered in compromised pregnancies. USDA-NRI grant 2007-01215 to LPR and ATGB; NIH grant HL64141 to LPR and DAR; ND EPSCoR AURA grant to ATGB and MAM; ND Space Grant Fellowship Program award to MAM; and NIH grant P20 RR016741 (INBRE program of the NCRR, NIH).