Figure 6.

Loss of muscle spindle-derived GDNF in the Egr3^KO and GDNF^FLOX/Egr3^CRE conditional mutant mouse. (A,B) GDNF (lacZ) is expressed in muscle spindles (black arrowheads) in P5 control gluteus maximus (GDNF^lacZ/-/Egr3^+/-) but absent in mutants (GDNF^lacZ/-/Egr3^KO). (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 GDNF^FLOX allele. (D) Southern blot analysis of genomic DNA from mouse tails. Wild-type (+/+) and GDNF^FLOX alleles are represented by 16 and 7.2 kb bands, respectively. (E) GDNF^FLOX/FLOX and GDNF^FLOX/+ 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 GDNF^FLOX/FLOX/Egr3^CRE/CRE mutant and GDNF^FLOX/FLOX/Egr3^WT 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 GDNF^FLOX/FLOX (no Cre) control (G) and in GDNF^FLOX/FLOX/Egr3^CRE/CRE animals (H). Scale bars: (B) 200 μm; (F), 50 μm; (H) 10 μm; (J) 50 μm.