Expression patterns of key Sonic Hedgehog signaling pathway components in the developing and adult mouse midbrain and in the MN9D cell line

Melanie Feuerstein,Christian Ophoven,Eleni Roussa,Konstantinos Michailidis,Shokoufeh Khakipoor,Enaam Chleilat

doi:10.1007/s00441-017-2664-2

Melanie Feuerstein, Christian Ophoven + Show 4 more

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https://doi.org/10.1007/s00441-017-2664-2

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Abstract

The temporal dynamic expression of Sonic Hedgehog (SHH) and signaling during early midbrain dopaminergic (mDA) neuron development is one of the key players in establishing mDA progenitor diversity. However, whether SHH signaling is also required during later developmental stages and in mature mDA neurons is less understood. We study the expression of SHH receptors Ptch1 and Gas1 (growth arrest-specific 1) and of the transcription factors Gli1, Gli2 and Gli3 in mouse midbrain during embryonic development [embryonic day (E) 12.5 onwards)], in newborn and adult mice using in situ hybridization and immunohistochemistry. Moreover, we examine the expression and regulation of dopaminergic neuronal progenitor markers, midbrain dopaminergic neuronal markers and markers of the SHH signaling pathway in undifferentiated and butyric acid-treated (differentiated) MN9D cells in the presence or absence of exogenous SHH in vitro by RT-PCR, immunoblotting and immunocytochemistry. Gli1 was expressed in the lateral mesencephalic domains, whereas Gli2 and Gli3 were expressed dorsolaterally and complemented by ventrolateral expression of Ptch1. Co-localization with tyrosine hydroxylase could not be observed. GAS1 was exclusively expressed in the dorsal mesencephalon at E11.5 and co-localized with Ki67. In contrast, MN9D cells expressed all the genes investigated and treatment of the cells with butyric acid significantly upregulated their expression. The results suggest that SHH is only indirectly involved in the differentiation and survival of mDA neurons and that the MN9D cell line is a valuable model for investigating early development but not the differentiation and survival of mDA neurons.

Highlights

Among the neurotransmitter systems in the brain, the midbrain dopaminergic system is of particular importance because of its relevance for several neuropsychiatric disorders
To elucidate the spatial and temporal expression of crucial Sonic Hedgehog (SHH) signaling components during mouse midbrain embryonic development, we first determined the expression of the SHH receptor Patched 1 (Ptch1), as well as of the transcription factors Gli1, Gli2 and Gli3 and established downstream determinants of the SHH signaling in mouse embryos at E12.5, E13.5 and E17.5 by in situ hybridization
In order to be able to examine whether Gli1, Gli2, Gli3 and Ptch1 are expressed in mDA neurons, we performed immunolabeling for TH on the same sections (Fig. 1)

Summary

Introduction

Among the neurotransmitter systems in the brain, the midbrain dopaminergic system is of particular importance because of its relevance for several neuropsychiatric disorders. Ventral midbrain dopaminergic (mDA) neurons play a pivotal role in several brain functions, among them voluntary movements, reward behaviors, cognition and emotions (Björklund and Dunnett 2007; Morales and Margolis 2017; Vogt Weisenhorn et al 2016). The molecular mechanisms underlying induction, differentiation and survival of mesencephalic dopaminergic neurons have been extensively studied. It is well established that induction of mDA neurons from progenitor cells depends on the temporal and spatial expression of patterning molecules, such as SHH and FGF8, which act as extrinsic determinants and of a subsequent activation of a combinatorial code of transcription factors representing the intrinsic determinants (reviewed in Smidt and Burbach 2007). During recent years, a molecular and functional heterogeneity has been experimentally identified, between neurons of A9 and A10

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