Development and initial characterization of xenomitochondrial mice.

Xenomitochondrial mice harboring trans-species mitochondria on a Mus musculus domesticus (MD) nuclear background were produced. We created xenomitochondrial ES cell cybrids by fusing Mus spretus (MS), Mus caroli (MC), Mus dunni (Mdu), or Mus pahari (MP) mitochondrial donor cytoplasts and rhodamine 6-G treated CC9.3.1 or PC4 ES cells. The selected donor backgrounds reflected increasing evolutionary divergence from MD mice and the resultant mitochondrial-nuclear mismatch targeted a graded respiratory chain defect. Homoplasmic (MS, MC, Mdu, and MP) and heteroplasmic (MC) cell lines were injected into MD ova, and liveborn chimeric mice were obtained (MS/MD 18 of 87, MC/MD 6 of 46, Mdu/MD 31 of 140, and MP/MD l of 9 founder chimeras, respectively). Seven MS/MD, 1 MC/MD, and 11 Mdu/MD chimeric founder females were mated with wild-type MD males, and 18 of 19 (95%) were fertile. Of fertile females, only one chimeric MS/MD (1% coat color chimerism) and four chimeric Mdu/MD females (80-90% coat color chimerism) produced homoplasmic offspring with low efficiency (7 of 135; 5%). Four male and three female offspring were homoplasmic for the introduced mitochondrial backgrounds. Three male and one female offspring proved viable. Generation of mouse lines using additional female ES cell lineages is underway. We hypothesize that these mice, when crossbred with neurodegenerative-disease mouse models, will show accelerated age-related neuronal loss, because of their suboptimal capacity for oxidative phosphorylation and putatively increased oxidative stress.

Developmental fate of mitochondria microinjected into murine zygotes.

A greater understanding of the fate of mitochondria injected into early preimplantation embryos would provide insights into mitochondrial biology and dynamics associated with development and disease. The ability to introduce foreign mitochondria into mouse embryos provides a means of tracking or following mitochondrial populations in vivo. Previously, injection of foreign mitochondria into the cytoplasm of the zygote was used to produce heteroplasmic mice. However, populations of introduced mitochondria decreased rapidly during development beyond the blastocyst stage. Therefore, the fate of exogenous mitochondria introduced into mouse ova was examined to determine viability and localization in comparison to endogenous mitochondria. Microinjection of murine mitochondria labeled with mitochondria-specific MitoTracker fluorophores allowed evaluation of subsequent viability and functionality of exogenous mitochondria populations in vivo. Characterization of mitochondrial survival and migration following microinjection illustrated toxic effects of MitoTracker Red upon exposure to laser confocal examination. In contrast, mitochondrial-specific fluorophores effectively detected foreign mitochondrial migration post-microinjection. The subsequent viability of the introduced mitochondria was observed through the blastocyst stage. Through the use of mitochondria-specific fluorophores, newly introduced mitochondria were further characterized and tracked post-transfer.

NBN defined medium supports the development of O4+/O1- immunopanned pro-oligodendroglia.

Maintenance of immunopanned cells in culture medium in the absence of serum or pre-conditioning by other neural cell types such as astrocytes can be problematic. Here we report the novel use of a chemically defined medium, which we refer to as NBN since it contains N-2 supplement, B-27 supplement, and N-acetyl-L-cysteine, for maintaining O4+/O1- immunopanned pro-oligodendroglia. Since we had previously characterized O4+/O1- immunopanned pro-oligodendroglia in astrocyte-conditioned basal defined medium (BDM; [24]), we compared their proliferation and differentiation in NBN medium or in NBN medium containing 40% NBN medium pre-conditioned by astrocytes. At 4 DIC in NBN, 23% of O4+ cells were BrdU+ while in conditioned NBN medium, 91% of O4+ cells were BrdU+. At 7 DIC in either medium, less than 25% of O4+ cells were BrdU+. O4+/O1- immunopanned pro-oligodendroglia cultured in NBN medium developed extensive processes and membranous expansions characteristic of mature oligodendroglia. At 4 DIC in NBN medium, approximately 100% of cells were O4+, 80% were O1+, and 54% were MBP+. By contrast, at 4 DIC in conditioned NBN, 87% of cells were O4+, 12% were O1+, and 2% were MBP+. At 7 DIC, there were no differences in the percentages of cells that expressed O4, O1, or MBP in either NBN or conditioned NBN. These results indicate that NBN defined medium supports the development of O4+/O1- immunopanned pro-oligodendroglia, and promotes more rapid maturation than conditioned NBN. The ability to maintain cells of the oligodendroglial lineage immunopanned at specific developmental stages in NBN defined medium should facilitate studies designed to identify effects of growth factors or toxins on oligodendroglia.

Development of O4+/O1- immunopanned pro-oligodendroglia in vitro.

In this study, O4+/O1- pro-oligodendroglia isolated by immunopanning from cerebral hemispheres of P3-P5 rats were evaluated during their maturation in culture. Immunopanning yielded 3-4 x 10(5) cells/cerebrum, with 98% O4+ and 6% O1+. There was heterogeneity in the morphologies of immunopanned cells ranging from simple bipolar cells to more complex multipolar cells. As a first step in determining potential differentiative responses of mature oligodendroglia, we examined glial fibrillary acidic protein (GFAP) expression in response to fetal bovine serum (FBS) by cultures established from O4+/O1- immunopanned cells grown for 1, 14, or 21 days, exposed to 20% FBS for 6-7 days and fixed and immunostained on days 7, 21 or 28 in culture (DIC). When immunopanned cells were exposed to FBS following 1 day in serum-free medium, 88% expressed GFAP and when immunopanned cells were cultured for 14 days prior to FBS exposure, 78% expressed GFAP. By contrast, when cells were cultured for 21 days prior to FBS exposure (when a majority of the cells expressed O1 and myelin basic protein (MBP)), only 19% of the cells expressed GFAP (p < 0.001). Cells that were O4+/GFAP- even in the presence of FBS often exhibited a mature oligodendroglial morphology. Among immunopanned cells that responded to FBS by expression of GFAP, both process-bearing (similar to type 2 astroglia) and flattened, polygonal (similar to type 1 astroglia) GFAP+ cells were observed. These results confirm the utility of immunopanning for the isolation of pro-oligodendroglia and demonstrate that oligodendroglia that develop in vitro from O4+/O1- immunopanned cells become resistant to GFAP induction by FBS.

Neural cell adhesion molecules modulate tyrosine phosphorylation of tubulin in nerve growth cone membranes.

Triggering neural cell adhesion molecules of the immunoglobulin superfamily with specific ligands or antibodies inhibited the phosphorylation of tryosyl residues in a subpopulation of alpha- and beta-tubulin associated with membranes from a subcellular fraction of nerve growth cones from fetal rat brain. Preincubation of these membranes with purified extracellular fragments of L1, N-CAM, or myelin-associated glycoprotein, or with antibodies directed against the extracellular domains of L1 or N-CAM, inhibited pp60c-src-dependent phosphorylation of tubulin in an endogenous membrane kinase reaction. Other proteins that affect neurite outgrowth (fibronectin, laminin, antibodies against N-cadherin) had no effect. The results suggest that cell adhesion molecules transduce cell surface events to intracellular signals by modulating the activity of protein tyrosine kinases or phosphatases in axonal membranes to influence cytoskeletal dynamics at the growth cone.

Cell type and developmental regulation of the fyn proto-oncogene in neural retina.

The product of the proto-oncogene c-fyn (p59fyn) is a non-receptor tyrosine kinase of unknown function. The expression of the p59fyn tyrosine kinase was analysed by immunoperoxidase staining of the different neuronal cell types in the developing chick neural retina. p59fyn was primarily localized in the cell bodies of mature retinal neurons. p59fyn immunoreactivity was most abundant in cell bodies of differentiated ganglion, amacrine and photoreceptor cells. The onset of p59fyn expression in developing photoreceptors occurred coordinately with terminal neuronal differentiation. p59fyn was also found within the outer plexiform layer, which contains synaptic terminals of the photoreceptors. At embryonic stages prior to photoreceptor differentiation, p59fyn was most highly concentrated in the cell bodies of differentiating ganglion and amacrine cells. p59fyn autokinase activity in retinal extracts decreased concomitant with the final stages of maturation of retinal neurons, suggesting that the p59fyn kinase is developmentally regulated. Thus, the expression of p59fyn is regulated in both a developmental and cell type-specific manner. The existence of p59fyn in some of the same neuronal cells as p60c-src suggests the possibility of functional redundancy of these non-receptor tyrosine kinases.

Tyrosine phosphorylated proteins decrease during differentiation of neuronal and glial cells.

Tyrosine kinases have been implicated in the development of the nervous system. To investigate their role, immunoblotting with phosphotyrosine antibodies was used to identify substrates of tyrosine kinases during glial and neuronal differentiation in the rat and mouse. Fourteen prominent phosphotyrosine-containing proteins were detected in oligodendrocyte-type 2 astrocyte (O2A) progenitor cells. When O2A cells differentiated into type 2 astrocytes, a phosphotyrosine-modified protein of 74 kilodaltons (kDa) decreased 15-fold in abundance, and phosphotyrosine-containing proteins of 36-40 kDa declined. When O2A cells differentiated into oligodendrocytes, a prominent 71-kDa phosphotyrosine-modified protein became undetectable. During retinoic acid-induced neuronal differentiation of the mouse embryonal carcinoma cell line P19S101A1 (P19), an 80-kDa phosphotyrosine-containing protein decreased from high levels in the undifferentiated state to undetectable levels after 96 h in aggregation. Retinoic acid treatment also caused a rapid decrease in levels of a 96-kDa phosphotyrosine-containing protein. Cell-cell contact occurring as a result of aggregation resulted in decreases in 130- and 90-kDa phosphotyrosine-containing proteins in both retinoic acid-induced and control cultures. Cultured rat central nervous system cerebral cortical neurons and peripheral nervous system dorsal root ganglion neurons exhibited prominent phosphotyrosine-modified proteins of 90 and 46 kDa the same sizes as those in P19 neurons. The phosphotyrosine-containing proteins involved in the retinoic acid-induced differentiation of P19 cells to neurons were different from those altered in the glial differentiation of O2A cells to astrocytes or oligodendrocytes, indicating that the tyrosine kinase substrates modified during nervous system differentiation may be cell-type-specific.

Expression of vimentin by cultured astroglia and oligodendroglia.

The objective of this study was to determine whether vimentin expression by process-bearing astroglia and oligodendroglia cultured from neonatal rat cerebral cortex resembled that in brain where vimentin is common in immature astroglia and a few subpopulations of mature astroglia, but is absent in oligodendroglia. Vimentin expression was detected by immunofluorescence microscopy using a monoclonal antibody (V9) against porcine lens vimentin in combination with either antiserum against the astroglial marker, glial fibrillary acidic protein (GFAP), or with antiserum against the oligodendroglial marker, galactocerebroside (GC). Specificity of the antivimentin antibody was indicated on immunoblots of process-bearing cell proteins separated by two-dimensional polyacrylamide gel electrophoresis. Enrichment of cultures for either GFAP+ astroglia or GC+ oligodendroglia was achieved by supplementation of the culture medium with fetal calf serum at 10% or 0.5%, respectively. Process-bearing cells maintained in 10% serum exhibited heterogeneity in their expression of GFAP and vimentin. Approximately half of the cells were GFAP+/vimentin+ throughout the 2-week culture period examined. GFAP+/vimentin- cells were a minor population at early times (3-4 days) in culture, but accounted for 40% of process-bearing cells after 2 weeks. Cultures maintained in reduced (0.5%) serum and stained for GC and vimentin also exhibited heterogeneity. Both GC+/vimentin+ and GC+/vimentin- cells were observed, with vimentin+ cells composing two-thirds and one-half of the GC+ population after 3 and 6 days, respectively, in reduced serum. The high incidence of vimentin expression by process-bearing astroglia and oligodendroglia suggests that these cultures contain glia in a relatively early stage of development.(ABSTRACT TRUNCATED AT 250 WORDS)

c-src and other proto-oncogenes implicated in neuronal differentiation.

The proto-oncogene c-src has been implicated in the development and mature function of the nervous system. pp60c-src, the protein product of the c-src gene, is a tyrosine protein kinase that is highly enriched in fetal neural tissue. pp60c-src appears in two phases of neuronal development. Neuroectodermal cells of gastrulating embryos first express pp60c-src around the time of commitment to neuronal or glial pathways. Later, committed neuroepithelial cells express pp60c-src near the onset of terminal neuronal differentiation. Immunocytochemical analyses of pp60c-src in developing chick retina, telencephalon, and cerebellum show immunoreactivity concentrated in regions rich in growth cones and neurites. Moreover, pp60c-src is concentrated approximately 10-fold in a biochemical fraction from fetal rat brain that is enriched in nerve growth cone membranes. These results point toward a function for pp60c-src in neurite outgrowth. A functional role for other proto-oncogenes in the development of the nervous system was indicated from a study of the expression of a battery of proto-oncogenes during the retinoic acid-induced differentiation of the mouse embryonal carcinoma cell line P19 to a neuronal phenotype. Nuclear runoff transcription of the proto-oncogenes c-src, c-fms, c-sis, N-ras, c-myc, and c-fos was observed in proliferating and retinoic acid-treated cells.

Plasticity of process-bearing glial cell cultures from neonatal rat cerebral cortical tissue.

The factors and cellular interactions that influence the commitment of cells to specific neural lineages are not well understood. We have used cultured non-neuronal process-bearing (PB) cells from neonatal rat cerebral cortices as a model to assess the influence of various culture conditions on the determination of cells as either astroglia or oligodendroglia. Increasing postseparation plating density was significantly associated (p less than 0.001) with decreasing percentages of glial fibrillary acidic protein (GFAP+) cells, increasing percentages of galactocerebroside (GC+) cells, and increasing percentages of nonstained cells. As the fetal calf serum content of growth medium was increased, the percentage of GFAP+ cells increased, and as the serum content was decreased, the percentage of GC+ cells increased. Evidence of minimal cell proliferation and the observation of PB cells that coexpressed GFAP and GC supported the conclusion that PB cells switched their phenotypic expression from GFAP+ in serum to GC+ in serum-free medium. PB cells exhibited plasticity in their phenotypic expression as cells grown for 9 d in serum-free medium were still responsive to the effects of serum, while cells grown for 6 d in serum were refractory to serum withdrawal. This research has demonstrated the plasticity of PB cells separated from polygonal astroglia as they expressed GFAP in the presence of serum and GC in serum-free medium.