Simultaneous Detection of Both GDNF and GFRα1 Expression Patterns in the Mouse Central Nervous System

Glial cell line-derived neurotrophic factor (GDNF) is proposed as a therapeutic tool in Parkinson’s disease, addiction-related disorders, and neurodegenerative conditions affecting motor neurons (MNs). Despite the high amount of work about GDNF therapeutic application, the neuronal circuits requiring GDNF trophic support in the brain and spinal cord (SC) are poorly characterized. Here, we defined GDNF and GDNF family receptor-α 1 (GFRα1) expression pattern in the brain and SC of newborn and adult mice. We performed systematic and simultaneous detection of EGFP and LacZ expressing alleles in reporter mice and asked whether modifications of this signaling pathway lead to a significant central nervous system (CNS) alteration. GFRα1 was predominantly expressed by neurons but also by an unexpected population of non-neuronal cells. GFRα1 expression pattern was wider in neonatal than in adult CNS and GDNF expression was restricted in comparison with GFRα1 at both developmental time points. The use of confocal microscopy to imaging X-gal deposits and EGFP allowed us to identify regions containing cells that expressed both proteins and to discriminate between auto and non-autotrophic signaling. We also suggested long-range GDNF-GFRα1 circuits taking advantage of the ability of the EGFP genetically encoded reporter to label long distance projecting axons. The complete elimination of either the ligand or the receptor during development did not produce major abnormalities, suggesting a preponderant role for GDNF signaling during adulthood. In the SC, our results pointed to local modulatory interneurons as the main target of GDNF produced by Clarke’s column (CC) cells. Our work increases the understanding on how GDNF signals in the CNS and establish a crucial framework for posterior studies addressing either the biological role of GDNF or the optimization of trophic factor-based therapies.


Supplementary Figure 2. Perinatal expression of GFRα1 and GDNF. Coronal brain sections
from Gfrα1 -(Egfp)/+ ; Gdnf -(LacZ)/+ mice brain showing GFRα1 (anti-EGFP, green) and GDNF expression (X-gal positive, blue areas drawn over left hemispheres). Scale bar represents 2.5 mm. Abbreviations are listed in Supp. Table 4. Distance from the most anterior part of the brain (mm) is shown.

Supplementary Figure 3. GDNF expressing areas in Gdnf-Egfp perinatal mouse.
Immunostaining anti-EGFP (green) in coronal brain sections (50 µm thickness) from P0 Gdnf-Egfp mouse. Scale bar represents 1.2 mm. Distance from the most anterior part of the brain is indicated (mm). Abbreviations are listed in Supp. Table 4.

Supplementary Tables
Supplementary Table 1
Description of expression patterns.
Description of expression pattern.

Supplementary Table 3. GFRα1 and GDNF expression in adult (P30) mouse brain
The study is based on at least three brains analyzed per structure. 1 Semi-quantitative analysis of the expression of GFRα1 and GDNF. −: None GFRα1/GDNF positive cell found; +: 1-20 GFRα1 positive / 1-5 GDNF positive; ++: 20-50 GFRα1 positive / 5-20 GDNF positive; and +++: more than 50 GFRα1 positive / more than 20 GDNF positive cells/analyzed structure. Cells are classified as Neurons when expression of NeuN was detected. 2 Semi-quantitative analysis of the expression of GFRα1 and GDNF in projections identified by morphology. High levels, ++; low, +; and absence of signal, -.

Supplementary Table 4. GFRα1 and GDNF expression in neonatal (P0) mouse brain
The study is based on at least three brains analyzed per structure. 1 Semi-quantitative analysis of the expression of GFRα1 and GDNF −: None GFRα1/GDNF positive cell found; +: 1-20 GFRα1 positive / 1-5 GDNF positive; ++: 20-50 GFRα1 positive / 5-20 GDNF positive; and +++: more than 50 GFRα1 positive / more than 20 GDNF positive cells/analyzed structure. 2 Semi-quantitative analysis of the expression of GFRα1 and GDNF in projections identified by morphology. High levels, ++; low, +; and absence of signal, -. 3 Discrepancies between LacZ and EGFP reporter mice are shown separated by a slash. 4 References where expression of GFRα1 and GDNF has been reported. 1: Golden et al. 1999;2: Hidalgo-Figueroa et al. 2012. 5 Concordance with Allen Brain Atlas GFRα1 in situ hybridization data. +: detected area; -: non detected area. n.a. not applicable. n.i.: structure not identified in the Allen Brain Atlas.