J. Douglas Coffin

Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation.

Basic fibroblast growth factor (FGF-2), an important modulator of cartilage and bone growth and differentiation, is expressed and regulated in osteoblastic cells. To investigate the role of FGF-2 in bone, we examined mice with a disruption of the Fgf2 gene. Measurement of trabecular bone architecture of the femoral metaphysis of Fgf2(+/+) and Fgf2(-/-) adult mice by micro-CT revealed that the platelike trabecular structures were markedly reduced and many of the connecting rods of trabecular bone were lost in the Fgf2(-/-) mice. Dynamic histomorphometry confirmed a significant decrease in trabecular bone volume, mineral apposition, and bone formation rates. In addition, there was a profound decreased mineralization of bone marrow stromal cultures from Fgf2(-/-) mice. This study provides strong evidence that FGF-2 helps determine bone mass as well as bone formation.

Changes in cerebral cortex size are governed by fibroblast growth factor during embryogenesis

We show that fibroblast growth factor 2 (FGF2) and FGF receptors are transiently expressed by cells of the pseudostratified ventricular epithelium (PVE) during early neurogenesis. A single microinjection of FGF2 into cerebral ventricles of rat embryos at E15.5 increased the volume and total number of neurons in the adult cerebral cortex by 18% and 87%, respectively. Microinjection of FGF2 by the end of neurogenesis, at E20.5, selectively increased the number of glia. Mice lacking the FGF2 gene had fewer cortical neurons and glia at maturity. BrdU studies in FGF2-microinjected and FGF2-null animals suggested that FGF2 increases the proportion of dividing cells in the PVE without affecting the cell-cycle length. Thus, FGF2 increases the number of rounds of division of cortical progenitors.

Fibroblast growth factor 2 control of vascular tone

Vascular tone control is essential in blood pressure regulation, shock, ischemia-reperfusion, inflammation, vessel injury/repair, wound healing, temperature regulation, digestion, exercise physiology, and metabolism. Here we show that a well-known growth factor, FCF2, long thought to be involved in many developmental and homeostatic processes, including growth of the tissue layers of vessel walls, functions in vascular tone control. Fgf2 knockout mice are morphologically normal and display decreased vascular smooth muscle contractility, low blood pressure and thrombocytosis. Following intra-arterial mechanical injury, FGF2-deficient vessels undergo a normal hyperplastic response. These results force us to reconsider the function of FGF2 in vascular development and homeostasis in terms of vascular tone control.

Absence of the tight junctional protein AF-6 disrupts epithelial cell–cell junctions and cell polarity during mouse development

BACKGROUND The establishment, maintenance and rearrangement of junctions between epithelial cells are extremely important in many developmental, physiological and pathological processes. AF-6 is a putative Ras effector; it is also a component of tight and adherens junctions, and has been shown to bind both Ras and the tight-junction protein ZO-1. In the mouse, AF-6 is encoded by the Af6 gene. As cell-cell junctions are important in morphogenesis, we generated a null mutation in the murine Af6 locus to test the hypothesis that lack of AF-6 function would cause epithelial abnormalities. RESULTS Although cell-cell junctions are thought to be important in early embryogenesis, homozygous mutant embryos were morphologically indistinguishable from wild-type embryos through 6.5 days post coitum (dpc) and were able to establish all three germ layers. The earliest morphological abnormalities were observed in the embryonic ectoderm of mutant embryos at 7.5 dpc. The length of the most apical cell-cell junctions was reduced, and basolateral surfaces of those cells were separated by multiple gaps. Cells of the embryonic ectoderm were less polarized as assessed by histological criteria and lateral localization of an apical marker. Mutant embryos died by 10 dpc, probably as a result of placental failure. CONCLUSIONS AF-6 is a critical regulator of cell-cell junctions during mouse development. The loss of neuroepithelial polarity in mutants is consistent with a loss of efficacy of the cell-cell junctions that have a critical role in establishing apical/basolateral asymmetry.

Angioblast differentiation and morphogenesis of the vascular endothelium in the mouse embryo

Bandeiraea simplicifolia B4 isolectin (BSLB4) and polyclonal antisera against von Willebrand factor (VWF) were used to study the origin of endothelial cells and their organization into blood vessels in the postimplantation mouse embryo. Examination of BSLB4-stained whole mounted and sectioned embryos revealed intense staining of the endothelium, highlighting large vessels, capillaries, and many individual cells. Dorsal aorta formation was first obvious at E7 when many lectin-positive cells appeared in paraxial and lateral plate mesoderm. As development proceeded to E8, BSLB4-positive cells became organized into craniocaudal lines destined to become the aorta proper. At E9, BSLB4 stained all vessels of the embryo including the dorsal aorta, the intersomitic arteries, and the endocardium. VWF expression was not detected until E8 when BSLB4/VWF double-stained sections revealed the dorsal aortae as the first VWF-positive vessels, while other endothelium visible with BSLB4 remained negative for VWF immunostaining. By E12 many other vessels became VWF-positive, including the aortic arches, the intersomitic arteries, and the cardinal veins. However, many angioblasts and capillaries remained VWF-negative, reflecting the heterogeneous expression of VWF among endothelium that has been reported in adults of other species. The histochemical data reported here support the conclusions of earlier avian studies by showing distinct vascular patterns in the initial formation of vessels from isolated angioblasts (vasculogenesis), followed by the extension and organization of the initial vascular structures (angiogenesis). Moreover, our data suggest that the endothelium arises from distinct VWF-positive sources associated with the dorsal aorta, as well as VWF-negative sources associated with other vessels in the embryo.

Disruption of the Fgf2 gene activates the adipogenic and suppresses the osteogenic program in mesenchymal marrow stromal stem cells

Here we determine the Fibroblast Growth Factor-2 (FGF2) dependency of the time course of changes in bone mass in female mice. This study extends our earlier reports that knockout of the FGF2 gene (Fgf2) caused low turnover bone loss in Fgf2(-/-) male mice by examining bone loss with age in Fgf2(-/-) female mice, and by assessing whether reduced bone formation is associated with differentiation of bone marrow stromal cells (BMSCs) towards the adipocyte lineage. Bone mineral density (BMD) was similar in 3-month-old female Fgf2(+/+) and Fgf2(-/-) mice but was significantly reduced as early as 5 months of age in Fgf2(-/-) mice. In vivo studies showed that there was a greater accumulation of marrow fat in long bones of 14 and 20 month old Fgf2(-/-) mice compared with Fgf2(+/+) littermates. To study the effect of disruption of FGF2 on osteoblastogenesis and adipogenesis, BMSCs from both genotypes were cultured in osteogenic or adipogenic media. Reduced alkaline phosphatase positive (ALP), mineralized colonies and a marked increase in adipocytes were observed in Fgf2(-/-) BMSC cultures. These cultures also showed an increase in the mRNA of the adipogenic transcription factor PPARgamma2 as well as the downstream target genes aP2 and adiponectin. Treatment with exogenous FGF2 blocked adipocyte formation and increased ALP colony formation and ALP activity in BMSC cultures of both genotypes. These results support an important role for endogenous FGF2 in osteoblast (OB) lineage determination. Alteration in FGF2 signaling may contribute to impaired OB bone formation capacity and to increased bone marrow fat accumulation both of which are characteristics of aged bone.

Immunohistologic labeling of murine endothelium

BACKGROUND Reliable identification of endothelial cells is a prerequisite for understanding vascularity changes in many cardiovascular diseases and therapeutic interventions. With the rising use of mouse models of disease and genetic manipulation, a consistent system to label murine endothelial cells in normal and diseased tissues would be an invaluable tool. METHODS Immunohistologic and histochemical methods were used to label endothelial cells in normal and infarcted heart as well as small intestine. Isolectin B(4) or antibodies to thrombomodulin, vWF, Tie-2, VE-cadherin, CD34, and CD31 were used to immunostain tissues fixed in either 4% formaldehyde (prepared fresh from powdered paraformaldehyde, PF), methyl Carnoys (MC), zinc (Zn) (all paraffin-embedded), or frozen sections. In addition, we used beta-galactosidase histochemistry in frozen sections from the Tie-2/beta-galactosidase transgenic mouse, in which the lacZ reporter gene is driven by the endothelial-specific Tie-2 promoter. RESULTS AND CONCLUSIONS Immunohistologic localization of endothelial cells was best accomplished using anti-CD31 in Zn-fixed, paraffin-embedded tissues. Antithrombomodulin staining also worked in Zn-fixed tissues as well as frozen tissues, but additional steps were required to reduce background. The beta-galactosidase reporter system also marked endothelial cells in frozen Tie-2 transgenic mouse tissues; however, this system required careful control of fixation and optimization of histochemistry and was not amenable to double labeling. Lectin staining gave complete labeling of endothelial cells but cross-reacted intensely with macrophages in the infarcted heart. We conclude that anti-CD31 immunostaining in Zn-fixed, paraffin-embedded murine tissue offered superior morphology and permitted optimal identification of proliferating endothelial cells during infarct repair.

Endogenous FGF-2 is critically important in PTH anabolic effects on bone.

Parathyroid hormone (PTH) increases fibroblast growth factor receptor‐1 (FGFR1) and fibroblast growth factor‐2 (FGF‐2) expression in osteoblasts and the anabolic response to PTH is reduced in Fgf2−/− mice. This study examined whether candidate factors implicated in the anabolic response to PTH were modulated in Fgf2−/− osteoblasts. PTH increased Runx‐2 protein expression in Fgf2+/+ but not Fgf2−/− osteoblasts. By immunocytochemistry, PTH treatment induced nuclear accumulation of Runx‐2 only in Fgf2+/+ osteoblasts. PTH and FGF‐2 regulate Runx‐2 via activation of the cAMP response element binding proteins (CREBs). Western blot time course studies showed that PTH increased phospho‐CREB within 15 min that was sustained for 24 h in Fgf2+/+ but had no effect in Fgf2−/− osteoblasts. Silencing of FGF‐2 in Fgf2+/+ osteoblasts blocked the stimulatory effect of PTH on Runx‐2 and CREBs phosphorylation. Studies of the effects of PTH on proteins involved in osteoblast precursor proliferation and apoptosis showed that PTH increased cyclinD1‐cdk4/6 protein in Fgf2+/+ but not Fgf2−/− osteoblasts. Interestingly, PTH increased the cell cycle inhibitor p21/waf1 in Fgf2−/− osteoblasts. PTH increased Bcl‐2/Bax protein ratio in Fgf2+/+ but not Fgf2−/− osteoblasts. In addition PTH increased cell viability in Fgf2+/+ but not Fgf2−/− osteoblasts. These data suggest that endogenous FGF‐2 is important in PTH effects on osteoblast proliferation, differentiation, and apoptosis. Reduced expression of these factors may contribute to the reduced anabolic response to PTH in the Fgf2−/− mice. Our results strongly indicate that the anabolic PTH effect is dependent in part on FGF‐2 expression. J. Cell. Physiol. 219: 143–151, 2009.

Nuclear Isoforms of Fibroblast Growth Factor 2 Are Novel Inducers of Hypophosphatemia via Modulation of FGF23 and KLOTHO

FGF2 transgenic mice were developed in which type I collagen regulatory sequences drive the nuclear high molecular weight FGF2 isoforms in osteoblasts (TgHMW). The phenotype of TgHMW mice included dwarfism, decreased bone mineral density (BMD), osteomalacia, and decreased serum phosphate (Pi). When TgHMW mice were fed a high Pi diet, BMD was increased, and dwarfism was partially reversed. The TgHMW phenotype was similar to mice overexpressing FGF23. Serum FGF23 was increased in TgHMW mice. Fgf23 mRNA in bones and fibroblast growth factor receptors 1c and 3c and Klotho mRNAs in kidneys were increased in TgHMW mice, whereas the renal Na+/Pi co-transporter Npt2a mRNA was decreased. Immunohistochemistry and Western blot analyses of TgHMW kidneys showed increased KLOTHO and decreased NPT2a protein. The results suggest that overexpression of HMW FGF2 increases FGF23/FGFR/KLOTHO signaling to down-regulate NPT2a, causing Pi wasting, osteomalacia, and decreased BMD. We assessed whether HMW FGF2 expression was altered in the Hyp mouse, a mouse homolog of the human disease X-linked hypophosphatemic rickets/osteomalacia. Fgf2 mRNA was increased in bones, and Western blots showed increased FGF2 protein in nuclear fractions from osteoblasts of Hyp mice. In addition, immunohistochemistry demonstrated co-localization of FGF23 and HMW FGF2 protein in osteoblasts and osteocytes from Hyp mice. This study reveals a novel mechanism of regulation of the FGF23-Pi homeostatic axis.

Lack of the Protein Tyrosine Phosphatase SHP-1 Results in Decreased Numbers of Glia Within the Motheaten (me/me) Mouse Brain

Mice that are homozygous for the autosomal recessive motheaten allele (me/me) lack the protein tyrosine phosphatase SHP‐1. Loss of SHP‐1 leads to many hematopoietic abnormalities, as well as defects such as infertility and low body weight. However, little is known regarding the role SHP‐1 plays in the development of the central nervous system (CNS). To define the role of SHP‐1 in CNS development and differentiation, we examined the brains of me/me mice at various times after birth for neuronal and glial abnormalities. Although the brains of me/me mice are slightly smaller than age‐matched wild‐type littermates, both me/me and wild‐type brains are similar in weight, possess an intact blood‐brain barrier, and have largely normal neuronal architecture. Significantly, the current study reveals that me/me brain shows decreases in the number of glial fibriallary acidic protein (GFAP)+ astrocytes and F480+ microglia compared with wild‐type mice. In addition, decreased immunostaining for the myelin‐synthesizing enzyme CNPase was observed in me/me mice, confirming the loss of myelin in these animals, as reported (Massa et al. [2000] Glia 29:376–385). It is particularly significant that there is a decreased number of immunolabeled glia of all subtypes and that this deficit in glial number is not restricted to a particular class of glia. This suggests that SHP‐1 is necessary for the normal differentiation and distribution of astrocytes, microglia, and oligendrocytes within the murine CNS. J. Comp. Neurol. 441:118–133, 2001.