In a screen of potential guidance cues that are expressed at the midline along the rostral course of mDN axons and temporally coincide with their passage (at E12.5–E15.5 in the mouse), we identified Shh as a candidate. Shh is expressed in the ventral midline (floor plate) at this time point in development, as demonstrated both by immunohistochemistry for Shh and in situ hybridization for Shh
mRNA transcript (). Prior studies have established the presence of a Shh gradient from ventral to dorsal regions throughout the neuroaxis, including the midbrain 
. mDN axons, identified by the expression of tyrosine hydroxylase (TH, the rate-limiting enzyme of dopamine synthesis), express the Shh receptor component Smoothened (Smo; ). Additionally, the Shh receptor component, Patched (Ptc), is expressed in the ventral midbrain region where the mDNn are located (). Interestingly, other canonical Shh signaling components including Gli1, Gli2, and Gli3 
; (data not shown), are not expressed in the mDNs at this stage of embryogenesis.
To investigate a role for Shh as a chemoattractant for mDN axons, we established an explant culture model in which E11.5 midbrain tissue was maintained next to an aggregate of HEK293 cells transfected with full-length Shh cDNA or control vector for 3 days (). Whereas control vector-transfected HEK293 had no effect, cells expressing Shh promoted unidirectional outgrowth toward the Shh source of TH-positive dopaminergic axons (). If Shh is a chemoattractant for TH-positive neurons, we would predict that ventral midline tissue rostral to the explant, which expresses Shh (), should also attract mDN axons. Consistent with this, ventral (but not dorsal) tissue was effective at attracting mDN axons (). Cyclopamine, a specific antagonist of Smo function, partially blocked the chemoattractant activity of ventral tissue, indicating a role for Shh as a ventral guidance cue for mDN axons. The remaining attractant activity of the ventral tissue is likely due to an additional, unidentified chemoattractant expressed in the rostral ventral areas. Shh did not alter the overall outgrowth of TH-positive axons from the explant, but rather promoted unidirectional outgrowth towards the chemoattractant (). As residual endogenous Shh may be present in the midbrain explants, the effects we observe may underestimate the role of Shh. E11.5 midbrain explants harbor mature mDNs, and consistent with this, Shh did not alter the number or specification of mDNs in the explants (data not shown).
Shh as a chemoattractant in mDN explant cultures.
To investigate the function of Shh in mDN axonal pathfinding in the intact CNS, we took advantage of conditional mutant mice in which the Shh receptor Smo was inactivated by a transgenic Cre recombinase regulated by the Nestin
cko mice). In these mice Cre-mediated recombination leads to complete inactivation of the Smo allele in the central nervous system by E11.5 
. Analysis of lateral dopaminergic fibers that project rostrally to the striatum in the Nestin-Smo
cko mice at E13.5 revealed normal projections (). In contrast, the medial dopaminergic projections that normally course ventromedially were defective in these mice (). This was particularly evident in the medial fibers that course most ventrally to extrastriatal targets including the pallidum and the subthalamus, as confirmed by immunostaining for the pallidum precursor marker Nkx2.1 (). Similar to the defects observed at E13.5 in Nestin-Smo
cko mice, the most medial projections remained defective at a later developmental time point, E15.5 (data not shown). These findings suggest a role for Shh signaling in the ventral targeting of medial dopaminergic axonal projections.
Smoothened conditional mutant mice display aberrant mDN axonal projections.
Dopaminergic projection alterations in Smoothened conditional mutant mice do not appear to be a consequence of target tissue defects.
Prior analyses and our expression studies argue strongly against the possibility that the defective mDN axonal targeting in Nestin-Smo
cko mice is an indirect effect of altered neuron specification of mDNs or target tissue. Patterning and specification of the embryonic ventral midbrain, including mDNs 
, as well as embryonic telencephalic target tissue such as the striatum 
, appear unaltered in Nestin-Smo
cko mice at the time period (E12.5–E16.5) during which the mDN axonal projections are formed ( and Figure S2
). The total density of mDN axonal projections appears unaltered in the mutant CNS (data not shown). Genetic fate mapping and conditional knockout analyses have demonstrated that canonical Shh signaling is no longer required in mDNs after E10 
. Consistent with this, gene expression of several markers for hypothalamic and forebrain target tissue, including FoxA2
, Islet 1
, and Shh
, appear normal in E12.5 Nestin-Smo
cko mice mice (). Expression of Netrin1, a chemoattractant present in the ventral midline, is similarly unaltered in the Nestin-Smo
cko mice ().
We hypothesized that lateral mDN axons, such as those that ascend to the striatum, are intrinsically less sensitive to Shh than the medial projections, and are thus less affected in the Nestin-Smo
cko mice. To test this, midbrain explants were divided into lateral and medial halves and then tested for chemoattraction to a Shh source (as described above). Axonal projections from medial (but not lateral) midbrain explants displayed unidirectional outgrowth towards the Shh point source (, Figure S3
). The molecular basis of this differential sensitivity remains to be determined; since the Shh receptor component Smo appears to be expressed evenly in both, medial and lateral mDN (data not shown).
Shh regulation of mDN axonal projections: dissection of signaling interactions.