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Gamma motor neurons express distinct genetic markers at birth and require muscle spindle-derived GDNF for postnatal survival.

Shneider NA, Brown MN, Smith CA, Pickel J, Alvarez FJ - Neural Dev (2009)

Bottom Line: Loss of muscle spindles also results in the downregulation of Gfralpha1 expression in some large diameter MNs, suggesting that spindle-derived factors may also influence populations of alpha-MNs with beta-skeletofusimotor collaterals.We also found that postnatal gamma-MNs are also distinguished by low expression of the neuronal nuclear protein (NeuN).Deletion of GDNF expression from muscle spindles results in the selective elimination of gamma-MNs with preservation of the spindle and its sensory innervation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Center for Motor Neuron Biology and Disease, Columbia University, New York, New York 10032, USA. ns327@columbia.edu

ABSTRACT

Background: Gamma motor neurons (gamma-MNs) selectively innervate muscle spindle intrafusal fibers and regulate their sensitivity to stretch. They constitute a distinct subpopulation that differs in morphology, physiology and connectivity from alpha-MNs, which innervate extrafusal muscle fibers and exert force. The mechanisms that control the differentiation of functionally distinct fusimotor neurons are unknown. Progress on this question has been limited by the absence of molecular markers to specifically distinguish and manipulate gamma-MNs. Recently, it was reported that early embryonic gamma-MN precursors are dependent on GDNF. Using this knowledge we characterized genetic strategies to label developing gamma-MNs based on GDNF receptor expression, showed their strict dependence for survival on muscle spindle-derived GDNF and generated an animal model in which gamma-MNs are selectively lost.

Results: In mice heterozygous for both the Hb9::GFP transgene and a tau-lacZ-labeled (TLZ) allele of the GDNF receptor Gfralpha1, we demonstrated that small motor neurons with high Gfralpha1-TLZ expression and lacking Hb9::GFP display structural and synaptic features of gamma-MNs and are selectively lost in mutants lacking target muscle spindles. Loss of muscle spindles also results in the downregulation of Gfralpha1 expression in some large diameter MNs, suggesting that spindle-derived factors may also influence populations of alpha-MNs with beta-skeletofusimotor collaterals. These molecular markers can be used to identify gamma-MNs from birth to the adult and to distinguish gamma- from beta-motor axons in the periphery. We also found that postnatal gamma-MNs are also distinguished by low expression of the neuronal nuclear protein (NeuN). With these markers of gamma-MN identity, we show after conditional elimination of GDNF from muscle spindles that the survival of gamma-MNs is selectively dependent on spindle-derived GDNF during the first 2 weeks of postnatal development.

Conclusion: Neonatal gamma-MNs display a unique molecular profile characterized by the differential expression of a series of markers - Gfralpha1, Hb9::GFP and NeuN - and the selective dependence on muscle spindle-derived GDNF. Deletion of GDNF expression from muscle spindles results in the selective elimination of gamma-MNs with preservation of the spindle and its sensory innervation. This provides a mouse model with which to explore the specific role of gamma-fusimotor activity in motor behaviors.

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Genetic elimination of muscle spindle-derived GDNF results in selective loss of gamma motor neurons. (A) Size distributions of ChAT+ MNs in GDNFFLOX/FLOX/Egr3WT (no Cre) controls at P20 are comparable to wild types (lines). (B) ChAT+ MNs losses in the absence of spindle-derived GDNF (GDNFFLOX/FLOX/Egr3CRE/CRE mutants). Small ChAT+ MNs represent 32 ± 1% ( ± SEM) of all MNs in GDNFFLOX/FLOX /Egr3+/+ and 16.8 ± 1.1% in GDNFFLOX/FLOX/Egr3CRE/CRE animals. Inset shows a depletion at P5 comparable to Egr3KO animals (see Figure 5F). (C) Similar loses in compound heterozygotes with one conditional and one  GDNF allele and a single copy of Egr3CRE (GDNFFLOX/LACZ/Egr3CRE/+). Inset shows a normal size distribution in one animal carrying one wild type and one floxed GDNF allele and a single Egr3CRE copy. (D) GDNF elimination from all muscle precursors using myf5CRE/+ results in similar losses of small ChAT+ MNs. Large MN numbers are unaffected in conditional mutants by targeted removal of GDNF from spindles. (E) Comparison of the percentage of small MNs (<480 μm2) in different genotypes. No differences were detected between wild-type and homozygous GDNFFLOX (no Cre) controls. Egr3KO mutants and several conditional/floxed GDNF mutants crossed to Egr3CRE or myf5CRE showed significant depletions compared to wild-types and GDNFFLOX (no Cre) controls (asterisks indicate P < 0.001 one-way ANOVA followed by P < 0.01 post-hoc Tukey comparisons). Depletion of small MNs in Egr3KO animals were more pronounced than in other genotypes, but differences were not statistically significant. N's, number of animals analyzed in each genotype.
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Figure 7: Genetic elimination of muscle spindle-derived GDNF results in selective loss of gamma motor neurons. (A) Size distributions of ChAT+ MNs in GDNFFLOX/FLOX/Egr3WT (no Cre) controls at P20 are comparable to wild types (lines). (B) ChAT+ MNs losses in the absence of spindle-derived GDNF (GDNFFLOX/FLOX/Egr3CRE/CRE mutants). Small ChAT+ MNs represent 32 ± 1% ( ± SEM) of all MNs in GDNFFLOX/FLOX /Egr3+/+ and 16.8 ± 1.1% in GDNFFLOX/FLOX/Egr3CRE/CRE animals. Inset shows a depletion at P5 comparable to Egr3KO animals (see Figure 5F). (C) Similar loses in compound heterozygotes with one conditional and one GDNF allele and a single copy of Egr3CRE (GDNFFLOX/LACZ/Egr3CRE/+). Inset shows a normal size distribution in one animal carrying one wild type and one floxed GDNF allele and a single Egr3CRE copy. (D) GDNF elimination from all muscle precursors using myf5CRE/+ results in similar losses of small ChAT+ MNs. Large MN numbers are unaffected in conditional mutants by targeted removal of GDNF from spindles. (E) Comparison of the percentage of small MNs (<480 μm2) in different genotypes. No differences were detected between wild-type and homozygous GDNFFLOX (no Cre) controls. Egr3KO mutants and several conditional/floxed GDNF mutants crossed to Egr3CRE or myf5CRE showed significant depletions compared to wild-types and GDNFFLOX (no Cre) controls (asterisks indicate P < 0.001 one-way ANOVA followed by P < 0.01 post-hoc Tukey comparisons). Depletion of small MNs in Egr3KO animals were more pronounced than in other genotypes, but differences were not statistically significant. N's, number of animals analyzed in each genotype.

Mentions: Size histograms of ChAT+ MNs revealed a normal bimodal distribution in P20 GDNFFLOX/FLOX controls (n = 3) and no significant difference in the calculated size average or SD of the small and large populations in these animals compared to the fitted distributions of age-matched wild-type animals analyzed previously (Figure 7A). In contrast, GDNFFLOX/FLOX/Egr3CRE/CRE animals (n = 5) showed a significant (approximately 50%; P < 0.001, t-test) decrease in the number of small (<485 μm2) ChAT+ MNs (Figure 7B, E). This reduction was smaller than, but statistically not different from, that observed in Egr3KO mutants (Figure 7E). Moreover, γ-MN depletion occurred with a similar time course in GDNFFLOX/FLOX/Egr3CRE/CRE animals (see inset in Figure 7B) compared to Egr3KO animals (Figure 5H). As a result, ChAT+ MNs in the GDNFFLOX/FLOX/Egr3CRE/CRE mutant comprise a single population well-fit by a single Gaussian (correlation = 0.86) with a mean average cross-sectional area of 708 μm2 ± 200 ( ± SD), which is similar to that estimated for the large MN population in GDNFFLOX/FLOX controls (758 ± 201 μm2). Similarly, selective loss of γ-MNs was also observed in mutants carrying a conditional (GDNFFLOX) and (GDNFLacZ) allele of GDNF and a single copy of Egr3CRE (Figure 7C, E).


Gamma motor neurons express distinct genetic markers at birth and require muscle spindle-derived GDNF for postnatal survival.

Shneider NA, Brown MN, Smith CA, Pickel J, Alvarez FJ - Neural Dev (2009)

Genetic elimination of muscle spindle-derived GDNF results in selective loss of gamma motor neurons. (A) Size distributions of ChAT+ MNs in GDNFFLOX/FLOX/Egr3WT (no Cre) controls at P20 are comparable to wild types (lines). (B) ChAT+ MNs losses in the absence of spindle-derived GDNF (GDNFFLOX/FLOX/Egr3CRE/CRE mutants). Small ChAT+ MNs represent 32 ± 1% ( ± SEM) of all MNs in GDNFFLOX/FLOX /Egr3+/+ and 16.8 ± 1.1% in GDNFFLOX/FLOX/Egr3CRE/CRE animals. Inset shows a depletion at P5 comparable to Egr3KO animals (see Figure 5F). (C) Similar loses in compound heterozygotes with one conditional and one  GDNF allele and a single copy of Egr3CRE (GDNFFLOX/LACZ/Egr3CRE/+). Inset shows a normal size distribution in one animal carrying one wild type and one floxed GDNF allele and a single Egr3CRE copy. (D) GDNF elimination from all muscle precursors using myf5CRE/+ results in similar losses of small ChAT+ MNs. Large MN numbers are unaffected in conditional mutants by targeted removal of GDNF from spindles. (E) Comparison of the percentage of small MNs (<480 μm2) in different genotypes. No differences were detected between wild-type and homozygous GDNFFLOX (no Cre) controls. Egr3KO mutants and several conditional/floxed GDNF mutants crossed to Egr3CRE or myf5CRE showed significant depletions compared to wild-types and GDNFFLOX (no Cre) controls (asterisks indicate P < 0.001 one-way ANOVA followed by P < 0.01 post-hoc Tukey comparisons). Depletion of small MNs in Egr3KO animals were more pronounced than in other genotypes, but differences were not statistically significant. N's, number of animals analyzed in each genotype.
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Figure 7: Genetic elimination of muscle spindle-derived GDNF results in selective loss of gamma motor neurons. (A) Size distributions of ChAT+ MNs in GDNFFLOX/FLOX/Egr3WT (no Cre) controls at P20 are comparable to wild types (lines). (B) ChAT+ MNs losses in the absence of spindle-derived GDNF (GDNFFLOX/FLOX/Egr3CRE/CRE mutants). Small ChAT+ MNs represent 32 ± 1% ( ± SEM) of all MNs in GDNFFLOX/FLOX /Egr3+/+ and 16.8 ± 1.1% in GDNFFLOX/FLOX/Egr3CRE/CRE animals. Inset shows a depletion at P5 comparable to Egr3KO animals (see Figure 5F). (C) Similar loses in compound heterozygotes with one conditional and one GDNF allele and a single copy of Egr3CRE (GDNFFLOX/LACZ/Egr3CRE/+). Inset shows a normal size distribution in one animal carrying one wild type and one floxed GDNF allele and a single Egr3CRE copy. (D) GDNF elimination from all muscle precursors using myf5CRE/+ results in similar losses of small ChAT+ MNs. Large MN numbers are unaffected in conditional mutants by targeted removal of GDNF from spindles. (E) Comparison of the percentage of small MNs (<480 μm2) in different genotypes. No differences were detected between wild-type and homozygous GDNFFLOX (no Cre) controls. Egr3KO mutants and several conditional/floxed GDNF mutants crossed to Egr3CRE or myf5CRE showed significant depletions compared to wild-types and GDNFFLOX (no Cre) controls (asterisks indicate P < 0.001 one-way ANOVA followed by P < 0.01 post-hoc Tukey comparisons). Depletion of small MNs in Egr3KO animals were more pronounced than in other genotypes, but differences were not statistically significant. N's, number of animals analyzed in each genotype.
Mentions: Size histograms of ChAT+ MNs revealed a normal bimodal distribution in P20 GDNFFLOX/FLOX controls (n = 3) and no significant difference in the calculated size average or SD of the small and large populations in these animals compared to the fitted distributions of age-matched wild-type animals analyzed previously (Figure 7A). In contrast, GDNFFLOX/FLOX/Egr3CRE/CRE animals (n = 5) showed a significant (approximately 50%; P < 0.001, t-test) decrease in the number of small (<485 μm2) ChAT+ MNs (Figure 7B, E). This reduction was smaller than, but statistically not different from, that observed in Egr3KO mutants (Figure 7E). Moreover, γ-MN depletion occurred with a similar time course in GDNFFLOX/FLOX/Egr3CRE/CRE animals (see inset in Figure 7B) compared to Egr3KO animals (Figure 5H). As a result, ChAT+ MNs in the GDNFFLOX/FLOX/Egr3CRE/CRE mutant comprise a single population well-fit by a single Gaussian (correlation = 0.86) with a mean average cross-sectional area of 708 μm2 ± 200 ( ± SD), which is similar to that estimated for the large MN population in GDNFFLOX/FLOX controls (758 ± 201 μm2). Similarly, selective loss of γ-MNs was also observed in mutants carrying a conditional (GDNFFLOX) and (GDNFLacZ) allele of GDNF and a single copy of Egr3CRE (Figure 7C, E).

Bottom Line: Loss of muscle spindles also results in the downregulation of Gfralpha1 expression in some large diameter MNs, suggesting that spindle-derived factors may also influence populations of alpha-MNs with beta-skeletofusimotor collaterals.We also found that postnatal gamma-MNs are also distinguished by low expression of the neuronal nuclear protein (NeuN).Deletion of GDNF expression from muscle spindles results in the selective elimination of gamma-MNs with preservation of the spindle and its sensory innervation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, Center for Motor Neuron Biology and Disease, Columbia University, New York, New York 10032, USA. ns327@columbia.edu

ABSTRACT

Background: Gamma motor neurons (gamma-MNs) selectively innervate muscle spindle intrafusal fibers and regulate their sensitivity to stretch. They constitute a distinct subpopulation that differs in morphology, physiology and connectivity from alpha-MNs, which innervate extrafusal muscle fibers and exert force. The mechanisms that control the differentiation of functionally distinct fusimotor neurons are unknown. Progress on this question has been limited by the absence of molecular markers to specifically distinguish and manipulate gamma-MNs. Recently, it was reported that early embryonic gamma-MN precursors are dependent on GDNF. Using this knowledge we characterized genetic strategies to label developing gamma-MNs based on GDNF receptor expression, showed their strict dependence for survival on muscle spindle-derived GDNF and generated an animal model in which gamma-MNs are selectively lost.

Results: In mice heterozygous for both the Hb9::GFP transgene and a tau-lacZ-labeled (TLZ) allele of the GDNF receptor Gfralpha1, we demonstrated that small motor neurons with high Gfralpha1-TLZ expression and lacking Hb9::GFP display structural and synaptic features of gamma-MNs and are selectively lost in mutants lacking target muscle spindles. Loss of muscle spindles also results in the downregulation of Gfralpha1 expression in some large diameter MNs, suggesting that spindle-derived factors may also influence populations of alpha-MNs with beta-skeletofusimotor collaterals. These molecular markers can be used to identify gamma-MNs from birth to the adult and to distinguish gamma- from beta-motor axons in the periphery. We also found that postnatal gamma-MNs are also distinguished by low expression of the neuronal nuclear protein (NeuN). With these markers of gamma-MN identity, we show after conditional elimination of GDNF from muscle spindles that the survival of gamma-MNs is selectively dependent on spindle-derived GDNF during the first 2 weeks of postnatal development.

Conclusion: Neonatal gamma-MNs display a unique molecular profile characterized by the differential expression of a series of markers - Gfralpha1, Hb9::GFP and NeuN - and the selective dependence on muscle spindle-derived GDNF. Deletion of GDNF expression from muscle spindles results in the selective elimination of gamma-MNs with preservation of the spindle and its sensory innervation. This provides a mouse model with which to explore the specific role of gamma-fusimotor activity in motor behaviors.

Show MeSH
Related in: MedlinePlus