Although many studies have been conducted on the development and function of the mDA system, the molecular mechanisms underlying induction, specification and survival of mDA neurons are still growing. The present work aimed to investigate the expression, both temporal and spatial, of the SHH signaling components during the development of mice mDA neurons at E12, E14 and E18. SHH is an early secreted morphogen by the floor plate and the notochord, which is involved in control of essential developmental processes, such as dorsoventral neural tube patterning, neural stem cell proliferation and cell fate specification . The critical involvement of SHH in the differentiation of mDA neurons has been controversially debated in the literatures.
Several previous studies had shown the critical role of SHH in the early development of mDA neurons. Cell fate mapping shows that mesencephalic cells expressing Gli1, present at the floor plate at E7.5-E9, are precursors to mDA neurons . Fate-mapping experiments clearly established that midbrain dopaminergic neurons are derived from precursors in the floor plate that express SHH, Foxa1/2 and Lmx1a/b cells [16, 20, 21]. Hynes and colleagues (1995) observed that SHH can induce dopaminergic neurons specification in vitro, so they suggested that the SHH is sufficient and necessary for induction of mDA neurons. Conversely, elimination of SHH signaling from the notochord by antibody blockade in vitro, or through gene targeting in mice (SHH mutant mice), prevents the differentiation of floor plate cells, the notochord degenerates, and motor neurons and most of ventral classes neurons of the neural tube are absent (dopaminergic and serotonergic neurons) [22, 23]. Using ISH with specific mRNA probes we showed that at E12 (Figs. 1 and 2) and E14 (Figs. 3 and 4) expression of Gli family and Ptch1 revealed a mosaic pattern in the mesencephalic domains. At E12, the genes studied were predominantly confined to the medial lateral mesencephalic domains, while at E14 Gli2 and Gli3 were appeared dorsolaterally and Ptch1 and Gli1expressed ventrolaterally. None of the studied genes was expressed in the same area of the mDA neurons (Figs. 2C, 4E and 4F).
Blaess and his colleagues reported that SHH plays a key role in the induction of a dopaminergic phenotype by modulating the FoxA2 expression via Gli1 between E8 and E8.5. They added that in response to SHH signalling; Gli1 expression was upregulated, which is a Gli2 dependent, whereas Gli3 is suppressed by SHH . The in vitro SHH treatment of E14 primary dissociated ventral mesencephalic cells significantly raised the number of surviving dopaminergic neurons . These previous results declared that SHH is necessary for induction and survival of dopaminergic neurons. In contrast, more recent reports have indicated that SHH inhibits progenitors to obtain a dopaminergic cell fate [26, 27]. In these studies, Wnt/β-catenin signaling is considered as the major molecular determinant responsible for mDA neuron development. Using conditionally knock-out mice and/or gene inducible fate mapping other studies have concluded that SHH signaling is important for the mDA development during the early developmental stages E8.5- E11, and acting as an early morphogen of the floor plate and ventral midbrain .
Tang and his team performed fate-mapping studies using conditional mutant mice that lacked Smoothened receptor in the ventral neurogenic niche for dopaminergic neurons during different embryonic ages at E8.5 and E10.5 . They observed that the removal of Smoothened at E8.5 lead to a significant loss in the DA at E10.5, while the removal of Smoothened at E10.5 lead to a transient loss at E12.5 and a sharp and continuous deficit was obvious in red nucleus, oculomotor and serotonergic neurons. Therefore, they suggested that SHH controls the development of the ventral midbrain neurons at the early embryonic stage.
Our results at E12 are in line with former observations of  and  at E11.5. A brilliant recent study analyzed the later embryonic stages and described different SHH signaling molecules . In our study we have extended the analysis of dynamic spatiotemporal changes of different SHH signaling molecules investigating their expression along the rostrocaudal axis. We observed that the expression pattern at E12 (Fig. 3) persisted until E14 (Fig. 4). Interestingly, at E18, neither expression of Gli3 nor Ptch1 was detectable at any mesencephalic domain (Fig. 6). Consequently, according to our investigation and these studies we could conclude that SHH is indirectly functining in the differentiation of mDA neurons.
Besides Patch1 and Cdon, both Boc, and Gas1 are identified as co-receptors that positively modulate SHH signalling activity. Gas1 and Boc play important and overlap ping roles during SHH patterning of the neural tube . Gas1 and Boc are required and cooperate in the promotion of SHH signaling during embryonic development, as assessed by loss-of-function experiments . Boc and Gas1 separately form discrete SHH receptor complexes with Ptch1 and are important for SHH-dependent cell proliferation. Cdon and Boc promote SHH-mediated cell fate specification and axon guidance. Among the function as tumor suppressor, Gas1 is involved in the development of the CNS being initially expressed in ventral progenitors of the spinal cord and in a recent study, expression of Gas1 has been detected in progenitors of the developing cortex and dentate gyrus .
The biological significance and requirement of Boc and Gas1 in SHH signalling has been highlighted in the work of , who reported that Gas1 and Boc interact with Ptch1 forming receptor complexes. Moreover, a SHH mutant protein capable to bind to Ptch1 but unable to bind Boc, Cdon or Gas1, hence, could not elicit SHH-dependent signaling and the cerebellar development was impaired in these mutants compared to control. Therefore, we concluded that there is absolute requirement for these co-receptors for the full activation of SHH signalling pathway. Based on these observations, we have studied distribution of Gas1 and Boc proteins in the mouse developing midbrain. At E14 Boc and Gas1 showed similar distribution, confined to the proliferative zone lining the aqueduct (Fig. 5), whereas, at E18 no expression could be detected for both (Fig. 6).
MN9D is a cell line commonly used to study differentiation processes of mDA. Therefore, we have asked whether expression of the genes of interest in this line corresponds to our in vivo situation (Fig. 7). MN9D cells expressed Ptch1, and Gli family, and exogenous SHH treatment of these cells significantly increased the expression of Gli1, Gli2 and SHH. Since cultured MN9D cells maybe heterogeneous regarding their differentiation state, i.e. some cells represent progenitors while others have already acquired a neuronal phenotype, we assumed that the expression of the above-mentioned molecules would be restricted to the undifferentiated cells. However, our data have surprisingly shown that both Ptch1, and Smoothened proteins were also abundant in MN9D cells, independently of their differentiation state. Moreover, Smoothened was expressed in TH expressing MN9D cells. These results clearly demonstrate that expression of SHH signaling molecules in MN9D cells do not match that of native mouse mesencephalic tissue. We observed that the mouse ventral midbrain primary cell culture form E14 ventral midbrain was expressing βIII- tubulin (a marker for mature neurons), but not Smoothened. Therefore, the primary culture results, in part, comparable to our in vivo data and more representative for in vivo studies.