Transplanted oligodendrocyte progenitor cells expressing a dominant-negative FGF receptor transgene fail to migrate in vivo

Abstract

The proliferation, migration, survival, and differentiation of oligodendrocyte progenitor cells, precursors to myelin-forming oligodendrocytes in the CNS, are controlled by a number of polypeptide growth factors in vitro. The requirement and roles for individual factors in vivo, however, are primarily unknown. We have used a cell transplantation approach to examine the role of fibroblast growth factor (FGF) in oligodendrocyte development in vivo. A dominant-negative version of the FGF receptor-1 transgene was introduced into oligodendrocyte progenitors in vitro, generating cells that were nonresponsive to FGF but responsive to other mitogens. When transplanted into the brains of neonatal rats, mutant cells were unable to migrate and remained within the ventricles. These results suggest a role for FGF signaling in establishing a motile phenotype for oligodendrocyte progenitor cell migration in vivo and illustrate the utility of a somatic cell mutagenesis approach for the study of gene function during CNS development in vivo.

Fig. 1.

Expression of a dominant-negative FGF receptor transgene in oligodendrocyte progenitor cells. A, Schematic representation of transfection vector pMo.FGFRx.iresNeo (pMoFRx), encoding (left toright) the Moloney LTR transcriptional regulatory sequence (Mo-LTR; arrow indicates polarity of transcription), a truncated form of the murine FGFR1 receptor (mFGFRx), internal ribosome entry sequence (IRES), and the neomycin phosphotransferase (NPTII) gene. The vertical arrowindicates the location of a stop codon introduced in FGFR1 byXbaI linker mutagenesis (Benvenisty and Ornitz, 1995) and causing premature termination of translation 22 amino acids after the transmembrane domain (tm) and upstream from the cytoplasmic tyrosine kinase (tk) domain.B, Anti-NPTII immunoreactivity in oligodendrocyte progenitor cells transfected with pMoFRx. Transfected cells had prominent staining of the soma. Scale bar, 25 μm. C,¹²⁵I-FGF binding proteins present in extracts of nontransfected parental progenitor cells (lane 1;wt) and progenitors transfected with pMoFRx (lane 2; FRx). Sizes of electrophoretic markers are indicated in kilodaltons, and arrows indicate a prominent FGF-binding protein migrating at the expected size for wild-type FGFR in both lanes and a protein with the predicted size of FGFRx (FRx; 100 kDa) in transfected cells.

Fig. 2.

Impaired FGF signaling in oligodendrocyte progenitor cells expressing the truncated mFGFRx receptor.A, B, [³H]Thymidine incorporation in nontransfected parental oligodendrocyte progenitor cells (squares) and two independently isolated, clonally derived strains transfected with pMoFRx (triangles,circles). Cells were cultured for 20 hr in the presence of the indicated concentrations of PDGF-AA (A) or FGF-2 (B), and results represent the mean ± SD of triplicate samples and are representative of a minimum of three independent assays. All cell strains responded to physiological levels of PDGF (5 ng/ml), although transfected cells were nonresponsive at physiological levels of FGF-2 (2–5 ng/ml).

Fig. 3.

Impaired migration of oligodendrocyte progenitor cells expressing the truncated mFGFRx receptor. Photomicrographs of parental oligodendrocyte progenitor cells (top) and progenitor cells transfected with pMoFRx (bottom) that were cultured for 72 hr in the presence of PDGF plus FGF-2 are shown. Parental progenitor cells showed extensive radial migration, whereas FGFRx cells did not. Short-term time lapse cinematography also indicated that transfected cells did not migrate (see Results). Scale bar, 650 μm.

Fig. 4.

Cotransplantation of fluorescent-labeled oligodendrocyte progenitor cells. Horizontal sections of postnatal day 4 rat brain (rostral, top) 48 hr after transplantation with parental progenitors (PKH2 label, greenfluorescence) plus pMoFRx-transfected progenitors (PKH26 label,red fluorescence). A, Retention of distinct fluorescent markers by cotransplanted wild-type and mutant cells. Transplanted cells located within the lateral ventricle (LV) specifically retained cell surface-associated PKH2 or PKH26 fluorescent markers. B, Migration of parental oligodendrocyte progenitor cells in host CNS. PKH2-labeled (wild-type) progenitor cells were found distal to the site of injection, entering the corpus callosum rostral to theLV. The fluorescence label was predominantly in the cell soma, whereas processes extended in situ in the presumed leading edge of migration (arrows) were less intensely labeled. Scale bar, 35 μm.

Fig. 5.

Transplanted FGFRx progenitor cells fail to migrate into brain parenchyma. Sagittal sections of transplant recipient rat brains 72 hr (A–C) and 7 d after transplantation (D); dorsal is to thetop and anterior is to the right for all micrographs. A, Phase micrograph showing the needle track through the cortex in the upper right(arrow depicts injection path). CC, Corpus callosum; LV, lateral ventricle;3V, third ventricle; and DG, dentate gyrus. B, Rhodamine fluorescence of PKH26-labeled FGFRx-expressing cells surrounding the dentate gyrus, delineated byboxed region in A. C, Region denoted by the arrow in B, showing FGFRx cells lining the hippocampal fissure and extending processes through the ependymal layer (arrows). D, Anti-NPTII immunoreactivity in FGFRx-expressing cells. Mutant cells found adjacent to the ependymal layer and the lateral ventricle continued to express the bicistronic NPTII transgene in vivo. CP, Choroid plexus. Scale bars:A, 650 μm; B, 120 μm;C, 30 μm; D, 25 μm.

Fig. 6.

Migration of control-transfected oligodendrocyte progenitor cells in vivo. Oligodendrocyte progenitor cells transfected with a nonsignaling (hCSFR1) receptor transgene migrate in rat brain. A, Anti-CSFR1 immunoreactivity of transfected O2A^fms progenitor cells in vitro. B, C, Horizontal sections of recipient brain 7 d after transplantation. Caudal istop; the same field is depicted in phase contrast (B) and fluorescence (C), showing PKH2-labeled O2A^fms cells that have migrated into parenchyma. Scale bar: A, 35 μm;B, 30 μm; C, 15 μm.

Fig. 7.

Quantitative analysis of the NPTII-immunoreactive transplanted cells in recipient rat brains. Serial sections from recipients of either pMoFRx-transfected progenitors (two independent lines; O2A^FRx; mean ± SD) or progenitor cells transfected with a control vector (O2A^PRα) were examined by NPTII immunohistochemistry at either 2 or 7 d after transplantation. Values represent the number of cells counted in each location relative to the total number of cells counted for each of the indicated time points. The periventricle was defined as the area within two cell diameters from the ventricular space. Mutant cells were found predominantly within the ventricles, whereas clonally derived control lines expressing NPTII were found within brain parenchyma over the same time course.