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Sonic hedgehog is a chemoattractant for midbrain dopaminergic axons.

Hammond R, Blaess S, Abeliovich A - PLoS ONE (2009)

Bottom Line: Finally, in mice in which Shh signaling is inactivated during late neuronal development, the most medial dopaminergic projections are deficient.In addition to the role of Shh in the induction of mDN precursors, Shh plays an important role in dopaminergic axon pathfinding to rostral target tissues.Furthermore, Shh signaling is involved in determining the structural diversity of these dopaminergic projections.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Center for Neurobiology and Behavior and Taub Institute, Columbia University College of Physicians and Surgeons, New York, New York, United States of America.

ABSTRACT
Midbrain dopaminergic axons project from the substantia nigra (SN) and the ventral tegmental area (VTA) to rostral target tissues, including the striatum, pallidum, and hypothalamus. The axons from the medially located VTA project primarily to more medial target tissues in the forebrain, whereas the more lateral SN axons project to lateral targets including the dorsolateral striatum. This structural diversity underlies the distinct functions of these pathways. Although a number of guidance cues have been implicated in the formation of the distinct axonal projections of the SN and VTA, the molecular basis of their diversity remains unclear. Here we investigate the molecular basis of structural diversity in mDN axonal projections. We find that Sonic Hedgehog (Shh) is expressed at a choice point in the course of the rostral dopaminergic projections. Furthermore, in midbrain explants, dopaminergic projections are attracted to a Shh source. Finally, in mice in which Shh signaling is inactivated during late neuronal development, the most medial dopaminergic projections are deficient. In addition to the role of Shh in the induction of mDN precursors, Shh plays an important role in dopaminergic axon pathfinding to rostral target tissues. Furthermore, Shh signaling is involved in determining the structural diversity of these dopaminergic projections.

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Related in: MedlinePlus

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.
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pone-0007007-g002: 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.

Mentions: 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 (Figure 2A). Whereas control vector-transfected HEK293 had no effect, cells expressing Shh promoted unidirectional outgrowth toward the Shh source of TH-positive dopaminergic axons (Figure 2B). If Shh is a chemoattractant for TH-positive neurons, we would predict that ventral midline tissue rostral to the explant, which expresses Shh (Figure 1A, 1C), should also attract mDN axons. Consistent with this, ventral (but not dorsal) tissue was effective at attracting mDN axons (Figure 2C). 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 (Figure 2B). 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).


Sonic hedgehog is a chemoattractant for midbrain dopaminergic axons.

Hammond R, Blaess S, Abeliovich A - PLoS ONE (2009)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2742719&req=5

pone-0007007-g002: 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.
Mentions: 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 (Figure 2A). Whereas control vector-transfected HEK293 had no effect, cells expressing Shh promoted unidirectional outgrowth toward the Shh source of TH-positive dopaminergic axons (Figure 2B). If Shh is a chemoattractant for TH-positive neurons, we would predict that ventral midline tissue rostral to the explant, which expresses Shh (Figure 1A, 1C), should also attract mDN axons. Consistent with this, ventral (but not dorsal) tissue was effective at attracting mDN axons (Figure 2C). 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 (Figure 2B). 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).

Bottom Line: Finally, in mice in which Shh signaling is inactivated during late neuronal development, the most medial dopaminergic projections are deficient.In addition to the role of Shh in the induction of mDN precursors, Shh plays an important role in dopaminergic axon pathfinding to rostral target tissues.Furthermore, Shh signaling is involved in determining the structural diversity of these dopaminergic projections.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Center for Neurobiology and Behavior and Taub Institute, Columbia University College of Physicians and Surgeons, New York, New York, United States of America.

ABSTRACT
Midbrain dopaminergic axons project from the substantia nigra (SN) and the ventral tegmental area (VTA) to rostral target tissues, including the striatum, pallidum, and hypothalamus. The axons from the medially located VTA project primarily to more medial target tissues in the forebrain, whereas the more lateral SN axons project to lateral targets including the dorsolateral striatum. This structural diversity underlies the distinct functions of these pathways. Although a number of guidance cues have been implicated in the formation of the distinct axonal projections of the SN and VTA, the molecular basis of their diversity remains unclear. Here we investigate the molecular basis of structural diversity in mDN axonal projections. We find that Sonic Hedgehog (Shh) is expressed at a choice point in the course of the rostral dopaminergic projections. Furthermore, in midbrain explants, dopaminergic projections are attracted to a Shh source. Finally, in mice in which Shh signaling is inactivated during late neuronal development, the most medial dopaminergic projections are deficient. In addition to the role of Shh in the induction of mDN precursors, Shh plays an important role in dopaminergic axon pathfinding to rostral target tissues. Furthermore, Shh signaling is involved in determining the structural diversity of these dopaminergic projections.

Show MeSH
Related in: MedlinePlus