[P130]: Early specification of dopaminergic phenotype during ES cell differentiation

Understanding how lineage choices are made during embryonic stem (ES) cell differentiation is critical for harnessing strategies for controlled production of therapeutic somatic cell types for cell transplantation and pharmaceutical drug screens. The in vitro generation of dopaminergic neurons, the type of cells lost in Parkinson's disease patients' brains, requires the inductive molecules sonic hedgehog and FGF8, or an unknown stromal cell derived inducing activity (SDIA). However, the exact identity of the responding cells and the timing of inductive activity that specify a dopaminergic fate in neural stem/progenitors still remain elusive. Using ES cells carrying a neuroepithelial cell specific vital reporter (Sox1-GFP) and FACS purification of Sox1-GFP neural progenitors, we have investigated the temporal aspect of SDIA mediated dopaminergic neuron specification during ES cell differentiation. Our results establish that SDIA induces a dopaminergic neuron fate in nascent neural stem or progenitor cells at, or prior to, Sox1 expression and does not appear to have further instructive role or neurotrophic activity during late neuronal differentiation of neural precursors. Furthermore, we show that dopaminergic neurons could be produced efficiently in a monolayer differentiation paradigm independent of SDIA activity or exogenous signalling molecules. In this case, the competence for dopaminergic neuron differentiation is also established at the level of Sox1 expression. Dopaminergic neurons are specified early during mouse ES cell differentiation. The subtype specification seems to be tightly linked with the acquisition of a pan neuroectoderm fate.

Poster abstracts / Int. J. Devl Neuroscience 24 (2006)   is suggested since treatment of dorsal-neural progenitor cells, in culture, with fibroblast growth factor 2 (FGF-2) results in OLP induction. To ask if dorsal induction of OLPs can occur in vivo, and if FGF-2 could initiate an alternative pathway of regulation, we used in utero microinjection of FGF-2 into the lateral ventricles of mouse fetal forebrain. A single injection of FGF-2 at E13.5 resulted in expression of the OLP markers Olig2 and PDGFRα mRNA in dorsal forebrain ventricular and intermediate zones. However, FGF-2 did not induce dorsal expression of Shh, Patched1 or Nkx2.1, suggesting that Shh signaling was not involved in this FGF-2 mediated dorsal induction. These results demonstrate that the dorsal forebrain in vivo has the potential to generate OLPs in the presence of normal positional cues, and that this can be driven by FGF-2 independent of Shh signaling.

Institute for Stem Cell Research, UK
Several protocols exist for differentiating dopaminergic neurons from embryonic stem (ES) cells. However, the exact mechanism and timing of dopaminergic neuron specification during in vitro differentiation using different protocols is not known.
We have investigated the temporal aspect of dopaminergic neuron specification during ES cell differentiation. By using ES cells carrying a neuroepithelial cell specific GFP reporter (Sox1-GFP, Ying et al., 2003), we investigate the timing and dopaminergic neuron differentiation potential of FACS purified Sox1-GFP neural precursors generated by either monolayer differentiation or PA6 differentiation.
Differentiation in co-culture with PA6 stromal cells is an efficient way of generating dopaminergic neurons from ES cells resulting in approximately 25% of the neurons expressing TH. To establish when the PA6 stromal derived activity (SDIA) is active during ES cell differentiation, we sorted the Sox1-GFP expressing neural progenitor cells formed in co-culture with PA6 cells and subsequently differentiated them either on PA6 cells again or on PDL/laminin coated plastic. We obtained a similar number of dopaminergic neurons in both replating conditions as well as in replated non-FACS sorted control cultures. This result indicates that SDIA acts at or prior to Sox1 expression.
Dopaminergic neuron can be also generated during monolayer differentiation albeit at a lower frequency. Similar to the PA6 co-culture system, monolayer derived FACS sorted Sox1-GFP expressing neural progenitor cells give rise to TH expressing neurons at a comparable frequency independent of further differentiation conditions. Taken together, our results establish the time window for PA6 stromal derived activity during ES cell differentiation. SDIA promotes dopaminergic fate specification in neural progenitors at or prior to Sox1 expression and does not appear to have further instructive role or neurotrophic activity during neuronal differentiation of neural precursors. Furthermore, our work suggests that dopaminergic specification can occur efficiently independent of SDIA activity, also at the level of Sox1 neural progenitors.

Beijing Institute for Neuroscience and Beijing Center of Neural Regeneration & Repairing, Capital University of Medical Sciences, China
Glial cell line-derived neurotrophic factor (GDNF) is one of the most potent trophic factors identified for promoting survival and function of dopaminergic (DA) neurons in the midbrain. Ret, a member of the receptor tyrosine kinase (RTK) superfamily transduces GDNF signalling. Some studies have implicated GDNF and Ret signalling in mammalian central nervous system (CNS) development, however, the mechanisms underlying this signalling pathway in vivo have not yet been clearly defined. Here, we demonstrate the involvement of the Ret signal transduction pathway in the DA neuron development both in vitro and in vivo. Neural precursors isolated from rat embryonic mesencephalon (E13.5d) were infected with pLNCX2-Ret and single-strand phosphorothioate Ret-AS-ODN was injected into the amniotic cavity of rat embryos of E14d in utero and embryos were examined 2 days later. The effects of the Ret ODN injection were assessed by measures of dopamine transporter (DAT) gene expression, striatal DA content and tyrosine hydroxylase (TH) positive fibers counts in the striatum. The techniques of immunocytochemistry, RT-PCR, Western blot and HPLC were used for those detections. The TUNEL staining was used for testing the apoptosis in the substantia nigra (SN) of the embryos. The DAT gene was clearly induced in rat embryonic neural precursors that had been transfected with Ret. Temporary blockade of Ret expression in embryos using Ret antisense oligonucleotides (Ret-AS-ODN) in vivo led to reduced striatal DA content and a decrease of TH positive fibers in the striatum. Additionally, some DA neurons in the SN underwent apoptotic cell death following the Ret-AS-ODN treatment. The data in present study suggest that normal function of Ret is required in vivo for the maturation