Generation of tyrosine hydroxylase-producing neurons from precursors of the embryonic and adult forebrain

Abstract

We have explored the plastic ability of neuronal precursors to acquire different identities by manipulating their surrounding environment. Specifically, we sought to identify potential signals involved in the specification of forebrain dopaminergic neurons. Here we describe culture conditions under which tyrosine hydroxylase (TH) expression is induced in neuronal precursors, which were derived directly from the embryonic striatum and adult subependyma (SE) of the lateral ventricle or generated from multipotent forebrain stem cells. TH was successfully induced in all of these cell types by 24 hr exposure to basic fibroblast growth factor (FGF2) and glial cell conditioned media (CM). The greatest magnitude of the inductive action was on embryonic striatal precursors. Although FGF2 alone induced limited TH expression in striatal cells (1.1 +/- 0.2% of neurons), these actions were potentiated 17.5-fold (19.6 +/- 1.5% of neurons) when FGF2 was coadministered with B49 glial cell line CM. Of these TH-immunoreactive cells, approximately 15% incorporated bromodeoxyuridine (BrdU), indicating that they were newly generated, and 95% coexpressed the neurotransmitter GABA. To investigate whether precursors of the adult forebrain subependyma were competent to respond to the instructive actions of FGF2+CM, they were first labeled in vivo with a pulse of BrdU. Although none of the cells expressed TH in control, 0.2% of total cells showed TH immunoreactivity in FGF2+CM-treated cultures. Under these same conditions only, in vitro-generated precursors from epidermal growth factor-responsive stem cells exhibited TH expression in 10% of their total neuronal progeny. Regulation of neurotransmitter phenotype in forebrain neuronal precursors, by the synergistic action of FGF2 and glial-derived diffusible factors, may represent a first step in understanding how these cells are generated in the embryonic and adult brain and opens the prospect for their manipulation in vitro and in vivo for therapeutic use.

Fig. 1.

Growth factor treatment increases the number of tyrosine hydroxylase-immunoreactive cells in primary cultures derived from different brain regions. Dispersed cells derived from the ventral mesencephalon, striatum, and cortex were grown on poly-l-ornithine-coated glass coverslips in the presence of the indicated factors (FBS, 0.1%; EGF, 20 ng/ml; FGF2, 20 ng/ml). Cells were fixed after 1 DIV and processed for TH and BrdU immunocytochemistry, as described in Materials and Methods. Neurons were identified by their immunoreactivity to anti-β-tubulin. The TH-IR neurons were expressed as a percentage of the number of TH-IR neurons present in FGF2-treated cultures. In FGF2-treated cultures from the ventral mesencephalon, 1.86 ± 0.12% of the total DAPI-stained nuclei and 6.6 ± 0.4% of the total neurons were TH-IR, respectively. In FGF2-treated cultures from the striatum, 0.31 ± 0.06% of the total cells and 1.1 ± 0.2 of the total neurons were TH-IR. Cultures derived from the cortex and treated with FGF2 showed the fewest TH-IR cells: ∼0.06% of the total number of live cells and 0.2% of the total number of neurons. Results are mean ± SEM of experiments performed three times on independent culture preparations, each performed in duplicate. Because of the represented scale of the x-axes, the error bars do not appear in some of the histograms. The asterisk indicates the level of significance with respect to FGF2-treated cultures;p < 0.05.

Fig. 2.

Conditioned media from glial cells potentiates the actions of FGF2 on the number of tyrosine hydroxylase-immunoreactive cells in cultures of striatal neuronal precursors. Dissociated striatal cells (5 × 10⁵) were grown on poly-l-ornithine-coated glass coverslips under the indicated conditions (CM, 75%; FGF2, 20 ng/ml). Cells were fixed after a 24 hr culture period and processed for indirect immunocytochemistry for TH, as described in Materials and Methods. Because control culture contained between 0 and 8 TH-IR cells and to allow for multiple comparison, the number of TH-IR neurons present in FGF2-treated cultures was taken as 100% (1.1 ± 0.2 TH-IR of the total number of neurons). Results are the mean ± SEM of three independent experiments, each performed in duplicate. Because of the represented scale of the x-axes, the error bars do not appear in some of the histograms. The asterisk indicates the level of significance with respect to CM-treated cultures;p < 0.01.

Fig. 3.

The combination of FGF2 and CM induces TH immunoreactivity in striatal neuronal precursors. Dissociated cells (5 × 10⁵) of the E14 striatum were cultured on poly-l-ornithine-coated glass coverslips in serum-free medium without (A, B) or with (C–G) 75% of B49 glial cell line-conditioned media and FGF2 (20 ng/ml) for 24 hr (A–D, F, G) and 3 d (E). Two hours after plating, all culture wells received 1 μm BrdU (a marker for DNA synthesis) (Gratzner, 1982). Fixed cells were processed for dual immunocytochemistry (as described in Materials and Methods) for TH and BrdU (A–D), TH and GABA (F, G), or single TH immunocytochemistry (E). In control cultures (A, B) the newly generated cells that had incorporated BrdU do not express TH. In FGF2+CM-treated cultures (C, D), the arrow shows one example of a cell that had incorporated BrdU and expressed TH. After 3 DIV, TH-IR cells developed typical neuronal morphology (E).F, G, Example of a 24-hr-old TH-IR cell that also coexpressed the neurotransmitter GABA. Scale bar (shown inE): A–D, 40 μm: E, 25 μm: F, G, 10 μm.

Fig. 4.

CM and FGF2 cooperate synergistically in a dose-dependent manner to induce TH expression in striatal neuronal precursors. A, Striatal cells were cultured in the presence of 20 ng/ml FGF2 and increasing concentrations of CM for a period of 24 hr and processed for TH immunocytochemistry, as described in Materials and Methods. A fluorescent-field microscope with 40× objective was used to count the total number of TH-IR cells in the entire area of each coverslip. B, Dissociated cells were cultured in the presence of 75% CM and increasing concentrations of either FGF2 or FGF1, and then processed for TH immunocytochemistry and quantified as described in Materials and Methods. Data represent the mean ± SEM of experiments performed three times on independent culture preparations, with two replicates for each condition within the independent experiments.

Fig. 5.

GDNF does not mimic the actions of CM on the appearance of tyrosine hydroxylase-immunoreactive neurons in cultures of striatal cells. Striatal cells were plated at a density of 5 × 10⁵ cells per well on poly-l-ornithine-coated glass coverslips and cultured under the indicated conditions for 24 hr before being processed for TH immunocytochemistry. Similar to Figures 1 and 2, the number of TH-IR neurons was represented as a percentage of the FGF2-treated cultures in which 1.1 ± 0.2 of the total number of neurons are TH-IR (see Results). Results are mean ± SEM of four independent experiments, each performed in duplicate.

Fig. 6.

Regulation of TH and GABA expression in the striatal neuronal precursors. A, Total RNA was isolated from the harvested 1-d-old cells and assayed (20 μg of RNA per lane) for the expression levels of TH transcripts (1.8 kb) by Northern blot analysis (see Materials and Methods for description of probe used and the conditions of hybridization). B, Total RNA samples were extracted from 1- and 3-d-old cultures that had been treated as indicated. The relative abundance of TH, GAD₆₇, and β-actin transcripts was assessed by RT-PCR (see Materials and Methods for details about the primer sequences and the conditions of the RT-PCR).

Fig. 7.

Induction of TH expression in precursors derived from the adult subependyma. Constitutively proliferating cells in the subependyma (SE) of adult mice were first labeledin vivo by five intraperitoneal injections of BrdU (see Materials and Methods). To confirm the in vivo location of the constitutively proliferating cells, a group of mice were perfused, and the brains were cryoprotected and cryostat-sectioned. The 10 μm sections were processed for BrdU immunocytochemistry. A, B, Drawing and photomicrograph of coronal section through the striatum of adult mouse. B, Photomicrograph of BrdU-IR cells within the SE surrounding the lateral ventricle. The location of this photomicrograph is outlined by the dotted lines inA. C–F, The SE cells were dissected, enzymatically dispersed, suspended in complete medium with or without FGF2+CM, and plated in the absence of BrdU. After culture periods of 1 DIV (C, D) and 3 DIV (E, F), the cells were fixed and processed for dual-label indirect immunocytochemistry for TH and BrdU (as outlined in Materials and Methods). Photomicrographs are of newly generated cells that had incorporated BrdU in vivo (C, E) and were TH-IR (D, F) in culture treated with FGF2+CM.aca, Anterior commissure, anterior; cc, corpus callosum; CTX, cortex; LV, lateral ventricle; SE, subependyma; STR, striatum. Scale bar (shown in F):B, 70 μm; C–F, 15 μm.

Fig. 8.

CM+FGF2 induces TH expression in neuronal precursors derived from multipotential neural precursor cells. Seven-day-old neurosphere clones, grown as described in Materials and Methods, were collected and mechanically dissociated in defined medium. Dispersed cells were either plated on poly-l-ornithine-coated glass coverslips (adherent culture conditions, A–C) or replated into a 75 cm² tissue culture flask (suspension cultures,D) in complete medium supplemented with growth factors and CM. Culture periods are indicated for each photomicrograph. Fixed cells or clones were processed for TH immunocytochemistry, as described in Materials and Methods. TH-IR cells were observed only in cultures treated with FGF2+CM (A–D). A–C, Time course evolution of morphological characteristics acquired by the TH-IR neurons in FGF2+CM-treated cultures (see Results).D, Photomicrograph of 7-d-old TH-IR clones grown in suspension in the presence of EGF (20 ng/ml), FGF2 (20 ng/ml), and CM (75%) (see Results). Scale bar (shown in D): A, B, 20 μm; C, 50 μm; D, 30 μm.