Cis and trans RET signaling control the survival and central projection growth of rapidly adapting mechanoreceptors
- University of Pennsylvania, United States
- Max Planck Institute of Neurobiology, Germany
- Parkinson's Disease and Movement Disorder Center of Maryland, United States
Figures
Figure 1 with 3 supplements
P7 Gfra2 mutant mice show normal dorsal spinal cord (dSC) VGLUT1 staining and Gfra1 null mice display normal rapidly adapting (RA) mechanoreceptor central projections at E13.5.
(A–B) Anti-VGLUT1 immunostaining of P7 SC sections from Gfra2GFP/+ control (A) and Gfra2GFP/GFP null (B) mice. VGLUT1 staining labels presynaptic terminals of mechanosensory neurons, which are found …
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Figure 1—source data 1
VGLUT1 dSC staining and RA mechanoreceptor number in P7 Gfra2 mutants.
- https://doi.org/10.7554/eLife.06828.004
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Figure 1—source data 2
RA mechanoreceptor central projections and cell number in E13.5 Ret, Gfra1, Gfra2, and Nrtn mutants.
- https://doi.org/10.7554/eLife.06828.005
Figure 1—figure supplement 1
Expression of Ret, Gfras, and Gfls in developing spinal cord (SC) and DRG.
(A–J) In situ hybridization of mouse SC and DRG at E13.5 and E15.5 for Ret (A–B), Gfra1 (C–D), Gfra2 (E–F), Gdnf (G–H), and Nrtn (I–J). Ret is expressed in DRG neurons and motor neurons at E13.5 and …
Figure 1—figure supplement 2
Ret is required for the growth of RA mechanosensory third order central projections at E13.5.
(A) Schematic of development of RA mechanosensory central projections. RA mechanoreceptors grow central and peripheral axons soon after neurogenesis, generating first order branches (red color). …
Figure 1—figure supplement 3
Generation of Gfra1 conditional and null alleles.
(A) Schematic of generation of Gfra1 conditional and null alleles. See supplemental ‘Materials and methods’ for additional details. (B) Predicted peptide sequence of the truncated GFRa1 protein …
Figure 2
Gfra2 and Nrtn null mice show reduced RA mechanoreceptor central projections at E13.5.
(A–D) Anti-GFP immunostaining to visualize RA mechanosensory central projections in E13.5 dSC sections of Gfra2GFP/+;RetCFP/+ control (A), Gfra2GFP/GFP;RetCFP/+ mutant (B), Nrtn+/−;RetCFP/+ control …
Figure 3 with 1 supplement
Central projection growth deficit of Gfra2 null RA mechanoreceptors at E15.5.
(A–I) E15.5 Gfra2GFP/+;RetCreERT/+;RosaTdt DRG sections stained with anti-RET (A–C), anti-NTRK1 (D–F), and anti-GFP (G–I). (J) Quantification of percentage of Tdt+ DRG neurons which co-express RET …
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Figure 3—source data 1
RA mechanoreceptor central projections and cell number in E15.5 Gfra2mutants.
- https://doi.org/10.7554/eLife.06828.011
Figure 3—figure supplement 1
Generation of tandem RetCreERT;RosaTdt allele.
The Ret and Rosa loci are located ∼5 megabases apart on mouse chromosome 6. RetCreERT/+ mice were crossed to RosaTdt/Tdt mice to generate RetCreERT/+;RosaTdt/+ mice, which were crossed back to RosaTd…
Figure 4 with 1 supplement
Ret and Gfra2 null mice display different central projection and cell survival deficits at E18.5.
(A–H) SC sections and whole mount L4/L5 DRGs of Tdt labeled RA mechanoreceptor from E18.5 RetCreERT/+;RosaTdt control (A–B), RetCreERT/CreERT;RosaTdt mutant (C–D), Gfra2GFP/+;RetCreERT/+;RosaTdt …
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Figure 4—source data 1
RA mechanoreceptor central projections and cell number in E18.5 Ret, Gfra2, Gfra1, and Gfra1;Gfra2 mutants.
- https://doi.org/10.7554/eLife.06828.014
Figure 4—figure supplement 1
Gfra2 null RA mechanoreceptors retain phospho-S6 expression.
(A–B) Anti-GFP (green) and anti-phospho-S6 (red) staining of P0 Gfra2GFP/+ control (A) and Gfra2GFP/GFP null (B) DRG sections. (C) Quantification of percentage of GFP+ neurons which express …
Figure 5
Gfra1;Gfra2 double null mice phenocopy Ret mutants at E18.5.
(A–H) SC sections and whole mount L4/L5 DRGs of Tdt labeled RA mechanoreceptors from E18.5 Gfra1+/−;RetCreERT/+;RosaTdt control (A–B), Gfra1−/−;RetCreERT/+;RosaTdt mutant (C–D), Gfra1+/−; Gfra2GFP/+;…
Figure 6 with 1 supplement
Gfra1 is not upregulated in Gfra2 null RA mechanoreceptors.
(A–B) Double fluorescent in situ hybridization against GFP and Gfra1 on E14.5 Gfra2GFP/+ control (A) and Gfra2GFP/GFP null (B) DRG sections. (C) Quantification of percentage of GFP+ neurons which …
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Figure 6—source data 1
QPCR of Gfra1 in embryonic Gfra2 null DRGs.
- https://doi.org/10.7554/eLife.06828.018
Figure 6—figure supplement 1
Quantitative RT-PCR (QPCR) of Gfra1 in Gfra2 null DRGs.
QPCR for Gfra1 from cDNAs generated from E13.5, E15.5, and E18.5 Gfra2GFP/+ control and Gfra2GFP/GFP null DRGs. (A) ΔCT values (cycles to reach threshold for Gfra1 minus cycles to reach threshold …
Figure 7 with 1 supplement
RA mechanoreceptors utilize GFRa1 produced by neighboring neurons to respond to GDNF.
(A–P) DRG explants from Gfra2GFP/+ control (A–D), Gfra2GFP/GFP null (E–H), Gfra2GFP/+;Ntrk1−/− null (I–L), and Gfra2GFP/GFP;Ntrk1−/− double null (M–P) embryos grown for 1 day in vitro and stained …
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Figure 7—source data 1
Quantification of axonal growth in Ret mutant DRG explants.
- https://doi.org/10.7554/eLife.06828.021
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Figure 7—source data 2
GFP+ neuron number in Gfra2 null and Gfra2;Ntrk1 double null explants.
- https://doi.org/10.7554/eLife.06828.022
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Figure 7—source data 3
Densimetric measurements of GFRa1 in DRG cell extracts and supernatants.
- https://doi.org/10.7554/eLife.06828.023
Figure 7—figure supplement 1
RetCFP null DRG explants lose responsiveness to GFLs.
(A–H) DRG explants from E14.5 RetCFP/+ control (A–D) and RetCFP/CFP null (E–H) embryos grown for 1 day in vitro and stained with anti-GFP antibody. Explants were treated with NRTN (50 ng/ml), GDNF …
Figure 8 with 1 supplement
Dynamic expression of GDNF during development.
(A–D) X-Gal staining of E13.5 (A, C) and E15.5 (B, D) GdnfLacZ/+ DRG and SC sections (also see Figure 8—figure supplement 1). Arrows indicate dorsal roots, which express Gdnf at E15.5, but not …
Figure 8—figure supplement 1
Gdnf LacZ expression in DRGs at E13.5 and E15.5.
DRG sections with large, strongly LacZ+ neurons were observed at both E13.5 (A) and E15.5 (B). Note that there are many more LacZ+ DRG neurons at E15.5. Sections with such cells were observed in all …
