Limits...
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.

Show MeSH

Related in: MedlinePlus

Loss of muscle spindle-derived GDNF in the Egr3KO and GDNFFLOX/Egr3CRE conditional mutant mouse. (A,B) GDNF (lacZ) is expressed in muscle spindles (black arrowheads) in P5 control gluteus maximus (GDNFlacZ/-/Egr3+/-) but absent in mutants (GDNFlacZ/-/Egr3KO). (C) Conditional gene targeting; loxP sites were introduced in the targeting construct around the GDNF gene coding sequence (CDS) before exon 3. An FRT-flanked neomycin-resistance (Neo) expression cassette was inserted upstream of the 5' loxP site and excised by crossing to ACTB-FLPe mice [14] to generate the GDNFFLOX allele. (D) Southern blot analysis of genomic DNA from mouse tails. Wild-type (+/+) and GDNFFLOX alleles are represented by 16 and 7.2 kb bands, respectively. (E) GDNFFLOX/FLOX and GDNFFLOX/+ mice were identified by PCR using primers P1 and P2 (shown as arrowheads) that flank the 3' loxP inserted in the 3' untranslated region of the GDNF gene. (F) In situ hybridization of P5 GDNFFLOX/FLOX/Egr3CRE/CRE mutant and GDNFFLOX/FLOX/Egr3WT control hindlimb muscle spindles with probes for Egr3 and GDNF. Analysis was performed on 10 μm-thick contiguous cryosections to demonstrate co-expression of Egr3 and GDNF in control and lack of GDNF expression in mutant muscle spindles after Cre recombination. (G,H) Semithin (1 μm) sections showing that control (G) and mutant spindles (H) have the same number of intrafusal muscle fibers (indicated by numbered arrows). (I,J) PGP9.5-immunoreactive annulospiral endings are similar in P20 GDNFFLOX/FLOX (no Cre) control (G) and in GDNFFLOX/FLOX/Egr3CRE/CRE animals (H). Scale bars: (B) 200 μm; (F), 50 μm; (H) 10 μm; (J) 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2800842&req=5

Figure 6: Loss of muscle spindle-derived GDNF in the Egr3KO and GDNFFLOX/Egr3CRE conditional mutant mouse. (A,B) GDNF (lacZ) is expressed in muscle spindles (black arrowheads) in P5 control gluteus maximus (GDNFlacZ/-/Egr3+/-) but absent in mutants (GDNFlacZ/-/Egr3KO). (C) Conditional gene targeting; loxP sites were introduced in the targeting construct around the GDNF gene coding sequence (CDS) before exon 3. An FRT-flanked neomycin-resistance (Neo) expression cassette was inserted upstream of the 5' loxP site and excised by crossing to ACTB-FLPe mice [14] to generate the GDNFFLOX allele. (D) Southern blot analysis of genomic DNA from mouse tails. Wild-type (+/+) and GDNFFLOX alleles are represented by 16 and 7.2 kb bands, respectively. (E) GDNFFLOX/FLOX and GDNFFLOX/+ mice were identified by PCR using primers P1 and P2 (shown as arrowheads) that flank the 3' loxP inserted in the 3' untranslated region of the GDNF gene. (F) In situ hybridization of P5 GDNFFLOX/FLOX/Egr3CRE/CRE mutant and GDNFFLOX/FLOX/Egr3WT control hindlimb muscle spindles with probes for Egr3 and GDNF. Analysis was performed on 10 μm-thick contiguous cryosections to demonstrate co-expression of Egr3 and GDNF in control and lack of GDNF expression in mutant muscle spindles after Cre recombination. (G,H) Semithin (1 μm) sections showing that control (G) and mutant spindles (H) have the same number of intrafusal muscle fibers (indicated by numbered arrows). (I,J) PGP9.5-immunoreactive annulospiral endings are similar in P20 GDNFFLOX/FLOX (no Cre) control (G) and in GDNFFLOX/FLOX/Egr3CRE/CRE animals (H). Scale bars: (B) 200 μm; (F), 50 μm; (H) 10 μm; (J) 50 μm.

Mentions: To test whether the loss of spindle-derived GDNF could account for the selective loss of γ-MNs we observed in the muscle spindle mutants, we first analyzed GDNF expression in muscle spindles in Egr3KO and ErbB2Δ animals using a targeted allele of GDNF (GDNFLacZ) in which β-galactosidase (lacZ) expression replaces GDNF [21]. GDNFLacZ was crossed into the Egr3KO background, and hindlimb muscles analyzed at P5 for GDNF (lacZ) expression. Histochemical staining for β-galactosidase activity revealed an absence of GDNF in postnatal Egr3KO mutant spindles (Figure 6A, B), demonstrating that GDNF expression is dependent on Egr3 function in the program of muscle spindle differentiation. Similar results were found in the rudimentary spindles of ErbB2Δ mutants at P5. These findings provide indirect evidence that spindle-derived GDNF is required for γ-MN survival.


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)

Loss of muscle spindle-derived GDNF in the Egr3KO and GDNFFLOX/Egr3CRE conditional mutant mouse. (A,B) GDNF (lacZ) is expressed in muscle spindles (black arrowheads) in P5 control gluteus maximus (GDNFlacZ/-/Egr3+/-) but absent in mutants (GDNFlacZ/-/Egr3KO). (C) Conditional gene targeting; loxP sites were introduced in the targeting construct around the GDNF gene coding sequence (CDS) before exon 3. An FRT-flanked neomycin-resistance (Neo) expression cassette was inserted upstream of the 5' loxP site and excised by crossing to ACTB-FLPe mice [14] to generate the GDNFFLOX allele. (D) Southern blot analysis of genomic DNA from mouse tails. Wild-type (+/+) and GDNFFLOX alleles are represented by 16 and 7.2 kb bands, respectively. (E) GDNFFLOX/FLOX and GDNFFLOX/+ mice were identified by PCR using primers P1 and P2 (shown as arrowheads) that flank the 3' loxP inserted in the 3' untranslated region of the GDNF gene. (F) In situ hybridization of P5 GDNFFLOX/FLOX/Egr3CRE/CRE mutant and GDNFFLOX/FLOX/Egr3WT control hindlimb muscle spindles with probes for Egr3 and GDNF. Analysis was performed on 10 μm-thick contiguous cryosections to demonstrate co-expression of Egr3 and GDNF in control and lack of GDNF expression in mutant muscle spindles after Cre recombination. (G,H) Semithin (1 μm) sections showing that control (G) and mutant spindles (H) have the same number of intrafusal muscle fibers (indicated by numbered arrows). (I,J) PGP9.5-immunoreactive annulospiral endings are similar in P20 GDNFFLOX/FLOX (no Cre) control (G) and in GDNFFLOX/FLOX/Egr3CRE/CRE animals (H). Scale bars: (B) 200 μm; (F), 50 μm; (H) 10 μm; (J) 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2800842&req=5

Figure 6: Loss of muscle spindle-derived GDNF in the Egr3KO and GDNFFLOX/Egr3CRE conditional mutant mouse. (A,B) GDNF (lacZ) is expressed in muscle spindles (black arrowheads) in P5 control gluteus maximus (GDNFlacZ/-/Egr3+/-) but absent in mutants (GDNFlacZ/-/Egr3KO). (C) Conditional gene targeting; loxP sites were introduced in the targeting construct around the GDNF gene coding sequence (CDS) before exon 3. An FRT-flanked neomycin-resistance (Neo) expression cassette was inserted upstream of the 5' loxP site and excised by crossing to ACTB-FLPe mice [14] to generate the GDNFFLOX allele. (D) Southern blot analysis of genomic DNA from mouse tails. Wild-type (+/+) and GDNFFLOX alleles are represented by 16 and 7.2 kb bands, respectively. (E) GDNFFLOX/FLOX and GDNFFLOX/+ mice were identified by PCR using primers P1 and P2 (shown as arrowheads) that flank the 3' loxP inserted in the 3' untranslated region of the GDNF gene. (F) In situ hybridization of P5 GDNFFLOX/FLOX/Egr3CRE/CRE mutant and GDNFFLOX/FLOX/Egr3WT control hindlimb muscle spindles with probes for Egr3 and GDNF. Analysis was performed on 10 μm-thick contiguous cryosections to demonstrate co-expression of Egr3 and GDNF in control and lack of GDNF expression in mutant muscle spindles after Cre recombination. (G,H) Semithin (1 μm) sections showing that control (G) and mutant spindles (H) have the same number of intrafusal muscle fibers (indicated by numbered arrows). (I,J) PGP9.5-immunoreactive annulospiral endings are similar in P20 GDNFFLOX/FLOX (no Cre) control (G) and in GDNFFLOX/FLOX/Egr3CRE/CRE animals (H). Scale bars: (B) 200 μm; (F), 50 μm; (H) 10 μm; (J) 50 μm.
Mentions: To test whether the loss of spindle-derived GDNF could account for the selective loss of γ-MNs we observed in the muscle spindle mutants, we first analyzed GDNF expression in muscle spindles in Egr3KO and ErbB2Δ animals using a targeted allele of GDNF (GDNFLacZ) in which β-galactosidase (lacZ) expression replaces GDNF [21]. GDNFLacZ was crossed into the Egr3KO background, and hindlimb muscles analyzed at P5 for GDNF (lacZ) expression. Histochemical staining for β-galactosidase activity revealed an absence of GDNF in postnatal Egr3KO mutant spindles (Figure 6A, B), demonstrating that GDNF expression is dependent on Egr3 function in the program of muscle spindle differentiation. Similar results were found in the rudimentary spindles of ErbB2Δ mutants at P5. These findings provide indirect evidence that spindle-derived GDNF is required for γ-MN survival.

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