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Trophic and proliferative effects of Shh on motor neurons in embryonic spinal cord culture from wildtype and G93A SOD1 mice.

Ma X, Turnbull P, Peterson R, Turnbull J - BMC Neurosci (2013)

Bottom Line: Shh increased the percentage of ciliated motor neurons, especially in G93A SOD1 culture.Moreover, Shh enhanced cell survival and differentiation of motor neuron precursors in WT culture.Shh is neurotrophic to motor neurons and has mitogenic effects in WT and mSOD1 G93A culture in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, McMaster University, 1200 Main St West, Hamilton, ON L8N 3Z5, Canada. turnbull@mcmaster.ca.

ABSTRACT

Background: The developmental morphogen sonic hedgehog (Shh) may continue to play a trophic role in the support of terminally-differentiated motor neurons, of potential relevance to motor neuron disease. In addition, it may support the proliferation and differentiation of endogenous stem cells along motor neuronal lineages. As such, we have examined the trophic and proliferative effects of Shh supplementation or Shh antagonism in embryonic spinal cord cell cultures derived from wildtype or G93A SOD1 mice, a mouse model of amyotrophic lateral sclerosis.

Results: Shh supported survival, and stimulated growth of motor neurons, neurite outgrowth, and neurosphere formation in primary culture derived from both G93A SOD1 and WT mice. Shh increased the percentage of ciliated motor neurons, especially in G93A SOD1 culture. Shh-treated cultures showed increased neuronal proliferation compared to controls and especially cyclopamine treated cultures, from G93A SOD1 and WT mice. Moreover, Shh enhanced cell survival and differentiation of motor neuron precursors in WT culture.

Conclusions: Shh is neurotrophic to motor neurons and has mitogenic effects in WT and mSOD1 G93A culture in vitro.

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Related in: MedlinePlus

Shh has trophic effects on motor neurons. Representative microscopic images show SMI32+ cells from WT mice treated with Shh 250 ng/ml had larger somata and more arborization (A) compared to cyclopamine-treated cells (B). It was shown that the average number of SMI32+ cells per field after treated with 250 ng/ml Shh was significantly higher than that control and cyclopamine (Shh 11 ± 1 vs control 5 ± 0, ** p = 0.0001; Shh 11 ± 1 vs cyclopamine 3 ± 0, * p = 0.0001; cyclopamine 3 ± 0 vs control 5 ± 0, δ p=0.017; C).
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Figure 2: Shh has trophic effects on motor neurons. Representative microscopic images show SMI32+ cells from WT mice treated with Shh 250 ng/ml had larger somata and more arborization (A) compared to cyclopamine-treated cells (B). It was shown that the average number of SMI32+ cells per field after treated with 250 ng/ml Shh was significantly higher than that control and cyclopamine (Shh 11 ± 1 vs control 5 ± 0, ** p = 0.0001; Shh 11 ± 1 vs cyclopamine 3 ± 0, * p = 0.0001; cyclopamine 3 ± 0 vs control 5 ± 0, δ p=0.017; C).

Mentions: We then focused on the effects of Shh or cyclopamine on motor neuron morphology and number. We treated WT and mSOD cultures with Shh (250 or 500 ng/ml) or cyclopamine (5 μM or 10 μM) between d2 and d10 and then examined SMI32 staining. At this time in culture, SMI32+ cells had long, ramified processes that stained intensely. Groups of positive cells were also present in neurospheres. Cultures treated with Shh 250 or 500 ng/ml had greatly increased SMI32 staining compared to control and especially compared to cells treated with cyclopamine. This is illustrated in Figure 2 where SMI32 staining in cells treated with Shh 250 ng/ml (Figure 2A) are compared with those treated with cyclopamine 5 μM (Figure 2B). Shh-treated cells had larger somata and more arborization, while in cyclopamine-treated but not Shh-treated cultures, dying cells were seen. TUNEL staining confirmed these cells were dying from apoptosis (not shown).


Trophic and proliferative effects of Shh on motor neurons in embryonic spinal cord culture from wildtype and G93A SOD1 mice.

Ma X, Turnbull P, Peterson R, Turnbull J - BMC Neurosci (2013)

Shh has trophic effects on motor neurons. Representative microscopic images show SMI32+ cells from WT mice treated with Shh 250 ng/ml had larger somata and more arborization (A) compared to cyclopamine-treated cells (B). It was shown that the average number of SMI32+ cells per field after treated with 250 ng/ml Shh was significantly higher than that control and cyclopamine (Shh 11 ± 1 vs control 5 ± 0, ** p = 0.0001; Shh 11 ± 1 vs cyclopamine 3 ± 0, * p = 0.0001; cyclopamine 3 ± 0 vs control 5 ± 0, δ p=0.017; C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852546&req=5

Figure 2: Shh has trophic effects on motor neurons. Representative microscopic images show SMI32+ cells from WT mice treated with Shh 250 ng/ml had larger somata and more arborization (A) compared to cyclopamine-treated cells (B). It was shown that the average number of SMI32+ cells per field after treated with 250 ng/ml Shh was significantly higher than that control and cyclopamine (Shh 11 ± 1 vs control 5 ± 0, ** p = 0.0001; Shh 11 ± 1 vs cyclopamine 3 ± 0, * p = 0.0001; cyclopamine 3 ± 0 vs control 5 ± 0, δ p=0.017; C).
Mentions: We then focused on the effects of Shh or cyclopamine on motor neuron morphology and number. We treated WT and mSOD cultures with Shh (250 or 500 ng/ml) or cyclopamine (5 μM or 10 μM) between d2 and d10 and then examined SMI32 staining. At this time in culture, SMI32+ cells had long, ramified processes that stained intensely. Groups of positive cells were also present in neurospheres. Cultures treated with Shh 250 or 500 ng/ml had greatly increased SMI32 staining compared to control and especially compared to cells treated with cyclopamine. This is illustrated in Figure 2 where SMI32 staining in cells treated with Shh 250 ng/ml (Figure 2A) are compared with those treated with cyclopamine 5 μM (Figure 2B). Shh-treated cells had larger somata and more arborization, while in cyclopamine-treated but not Shh-treated cultures, dying cells were seen. TUNEL staining confirmed these cells were dying from apoptosis (not shown).

Bottom Line: Shh increased the percentage of ciliated motor neurons, especially in G93A SOD1 culture.Moreover, Shh enhanced cell survival and differentiation of motor neuron precursors in WT culture.Shh is neurotrophic to motor neurons and has mitogenic effects in WT and mSOD1 G93A culture in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, McMaster University, 1200 Main St West, Hamilton, ON L8N 3Z5, Canada. turnbull@mcmaster.ca.

ABSTRACT

Background: The developmental morphogen sonic hedgehog (Shh) may continue to play a trophic role in the support of terminally-differentiated motor neurons, of potential relevance to motor neuron disease. In addition, it may support the proliferation and differentiation of endogenous stem cells along motor neuronal lineages. As such, we have examined the trophic and proliferative effects of Shh supplementation or Shh antagonism in embryonic spinal cord cell cultures derived from wildtype or G93A SOD1 mice, a mouse model of amyotrophic lateral sclerosis.

Results: Shh supported survival, and stimulated growth of motor neurons, neurite outgrowth, and neurosphere formation in primary culture derived from both G93A SOD1 and WT mice. Shh increased the percentage of ciliated motor neurons, especially in G93A SOD1 culture. Shh-treated cultures showed increased neuronal proliferation compared to controls and especially cyclopamine treated cultures, from G93A SOD1 and WT mice. Moreover, Shh enhanced cell survival and differentiation of motor neuron precursors in WT culture.

Conclusions: Shh is neurotrophic to motor neurons and has mitogenic effects in WT and mSOD1 G93A culture in vitro.

Show MeSH
Related in: MedlinePlus