Zusammenfassung
Background
The ventral midbrain contains a diverse array of neurons, including dopaminergic neurons of the ventral tegmental area (VTA) and substantia nigra (SN) and neurons of the red nucleus (RN). Dopaminergic and RN neurons have been shown to arise from ventral mesencephalic precursors that express Sonic Hedgehog (Shh). However, Shh expression, which is initially confined to the mesencephalic ventral midline, expands laterally and is then downregulated in the ventral midline. In contrast, expression of the Hedgehog target gene Gli1 initiates in the ventral midline prior to Shh expression, but after the onset of Shh expression it is expressed in precursors lateral to Shh-positive cells. Given these dynamic gene expression patterns, Shh and Gli1 expression could delineate different progenitor populations at distinct embryonic time points.
Results
We employed genetic inducible fate mapping (GIFM) to investigate whether precursors that express Shh (Shh-GIFM) or transduce Shh signaling (Gli1-GIFM) at different time points give rise to different ventral midbrain cell types. We find that precursors restricted to the ventral midline are labeled at embryonic day (E)7.5 with Gli1-GIFM, and with Shh-GIFM at E8.5. These precursors give rise to all subtypes of midbrain dopaminergic neurons and the anterior RN. A broader domain of progenitors that includes the ventral midline is marked with Gli1-GIFM at E8.5 and with Shh-GIFM at E9.5; these fate-mapped cells also contribute to all midbrain dopaminergic subtypes and to the entire RN. In contrast, a lateral progenitor domain that is labeled with Gli1-GIFM at E9.5 and with Shh-GIFM at E11.5 has a markedly reduced potential to give rise to the RN and to SN dopaminergic neurons, and preferentially gives rise to the ventral-medial VTA. In addition, cells derived from Shh- and Gli1-expressing progenitors located outside of the ventral midline give rise to astrocytes.
Conclusions
We define a ventral midbrain precursor map based on the timing of Gli1 and Shh expression, and suggest that the diversity of midbrain dopaminergic neurons is at least partially determined during their precursor stage when their medial-lateral position, differential gene expression and the time when they leave the ventricular zone influence their fate decisions.
Background
The ventral mesencephalic progenitor domain generates a diverse array of distinct neuronal cell types, including neurons of the red nucleus (RN), motoneurons of the oculomotor nucleus and midbrain dopaminergic (DA) neurons. DA neurons are further organized into anatomically and functionally distinct subclasses [1]. The substantia nigra (SN), located in the lateral-ventral midbrain, projects to the dorsal-lateral striatum and is involved in the regulation of motor behaviors. The ventral tegmental area (VTA), located more medially, projects to corticolimbic targets and is important for motivational states. The retrorubral field is located posterior to the SN and projects to striatal, limbic and cortical areas. The functional diversity of these different regions becomes apparent in disease states: in Parkinson's disease, SN neurons, but not VTA neurons, degenerate, resulting in severe motor deficits. In contrast, abnormalities in the mesocorticolimbic system have been implicated in addiction, schizophrenia and attention deficit disorder [2–4]. While it is well established that the functional diversity of ventral midbrain neurons and DA subclasses is based on their distinct efferent and afferent connections and their distinct molecular make-up and physiology, it remains unclear when and how these distinct neuronal (sub)classes are established during development.
All midbrain DA neurons appear to arise from ventral mesencephalic floor plate progenitors that express Sonic Hedgehog (Shh) [5–8]. A recent paper utilizing genetic inducible fate mapping (GIFM) [9] suggested that Shh expression between embryonic day (E)7.5 and E12.5 sequentially marks three spatially distinct ventral mesencephalic progenitor domains that give rise to different neurons. However, the distribution of fate-mapped cells was only assessed qualitatively at embryonic stages, and a potential contribution to glia was not determined.
Gli1, a zinc finger transcription factor in the Shh signaling pathway, is only transcribed in cells that receive high levels of Hedgehog signaling (and are close to the source of Hedgehog) [10, 11]; therefore, its expression can be used as a readout for cells that are exposed to high levels of Shh signaling [12]. Shh-expressing cells, including the floor plate cells themselves, do not respond to Shh signaling as measured by the expression of Gli1 [11–13]. It is therefore necessary to understand the exact timing of Shh responses and Shh expression in ventral midbrain precursors to gain a better insight into the role of Shh signaling in specification of ventral midbrain neurons.
To establish a precise precursor map of the ventral mesencephalon, we assessed the fate of Gli1-expressing (Shh-responding) and Shh-expressing progenitors with GIFM in a quantitative manner at embryonic and postnatal stages. We show that Gli1 expression precedes Shh expression by about a day and demonstrate that ventral midbrain precursors that give rise to DA neurons respond to Shh signaling between E7.5 and E9.5 and express Shh between E8.5 and E11.5. Progenitors in the ventral midline that are labeled with Gli1-GIFM at E7.5 and with Shh-GIFM at E8.0 to E8.5 contribute to midbrain DA neurons and the anterior RN. Progenitors in a broader domain are marked with Gli1-GIFM at E8.5 and Shh-GIFM at E9.5 to E10.5 and show a strong contribution to all subsets of DA neurons and to RN neurons. Precursors adjacent to the ventral midline that are fate-mapped with Gli1-GIFM at E9.5 and Shh-GIFM at E11.5 maintain the potential to develop into DA neurons of the ventral-medial VTA. However, they contribute few cells to DA neurons in the SN and to RN neurons. In addition, precursors labeled with Gli1-GIFM at E8.5 to E9.5 give rise to other ventral midbrain neurons, including neurons in the oculomotor nucleus and the non-DA neurons in the SN reticularis, consistent with a broad medial-lateral distribution of Gli1-expressing precursors. Finally, we observe that Shh- and Gli1-expressing progenitors, with the exception of progenitors in the ventral midline, develop into ventral midbrain astrocytes.
