Sonic Hedgehog Is a Chemoattractant for Midbrain Dopaminergic Axons
Figure 2
Shh as a chemoattractant in mDN explant cultures.
(A) Schematic of explant dissection. The ventral third of the midbrain neuroepithelium, including the floor plate, of E11.5 mouse was dissected and placed in an ‘open-book’ preparation in apposition to transfected HEK293T cells or appropriate tissue as indicated. HB – hindbrain, MB – midbrain, FB – forebrain. (B) E11.5 bilateral ventral midbrain explants cultures (FP – floor plate) in apposition to mock-transfected or Shh transfected HEK293T cells (delineated by white dotted lines). Explants and cell clusters were cocultured for 3 days, fixed, and immunostained for TH. Explants demonstrated increased TH-positive axonal outgrowth in the rostral quadrant (facing the explant – quadrants delineated by yellow dotted lines) in the presence of a Shh source. This effect was diminished by the addition of cyclopamine (Cyc) at 10 nM to the culture medium. The axonal extension from the rostral side was quantified using pixel counting software (see Materials and Methods) and expressed as a percentage of total rostral and caudal outgrowth. The effect of Shh was significant when tested using the two-tailed Student T-test (p<0.05), n>25 explants in each condition. Total (non-directional) outgrowth from the lateral and caudal sides remained unaffected by the presence of Shh. (C) The effects of Shh transfected HEK293T cells can be mimicked by the presence of rostro-ventral neuroepithelium known to express Shh (excluding the zona limitans intrathalamica; ZLI), and this effect can be reduced by the addition of cyclopamine to the medium. This effect is significant (p<0.05) by the two-tailed Student T-test, n>25 explants in each condition. Total axonal outgrowth from the caudal and lateral sides of the explants appears unaffected by the presence of dorsal or ventral tissue pieces. However dorsal tissue, which lacks Shh expression, does appear to have a positive effect on dopaminergic axonal outgrowth from the rostral side of ventral midbrain explants, an effect that is not blocked by cyclopamine implicating a Shh/Smo independent mechanism. Scale bar: (B+C) 100 µm.