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Development of the urogenital system is regulated via the 3′UTR of GDNF

Mechanisms controlling ureter lenght and the position of the kidney are poorly understood. Glial cell-line derived neurotrophic factor (GDNF) induced RET signaling is critical for ureteric bud outgrowth, but the function of endogenous GDNF in further renal differentiation and urogenital system development remains discursive. Here we analyzed mice where 3′ untranslated region (UTR) of GDNF is replaced with sequence less responsive to microRNA-mediated regulation, leading to increased GDNF expression specifically in cells naturally transcribing Gdnf. We demonstrate that increased Gdnf leads to short ureters in kidneys located in an abnormally caudal position thus resembling human pelvic kidneys. High GDNF levels expand collecting ductal progenitors at the expense of ureteric trunk elongation and result in expanded tip and short trunk phenotype due to changes in cell cycle length and progenitor motility. MEK-inhibition rescues these defects suggesting that MAPK-activity mediates GDNF’s effects on progenitors. Moreover, Gdnf   hyper mice are infertile likely due to effects of excess GDNF on distal ureter remodeling. Our findings suggest that dysregulation of GDNF levels, for example via alterations in 3′UTR, may account for a subset of congenital anomalies of the kidney and urinary tract (CAKUT) and/or congenital infertility cases in humans and pave way to future studies.


Animals
All animal work was conducted according to European Union directives (Directive 2010/EU/63), as well as in compliance with the Code of Ethical Conduct for Animal Experimentation. The licenses for mouse lines used in this publication were KEK16-020 and ESAVI/11198/04.10.07/2014. Mice were housed in individually ventilated cages with optimal humidification and heating and maintained in the certified animal facilities. Cleaning, disinfection and general care is provided by the professionals and following mandatory federal and internal regulations.

Tissue processing
For hematoxylin-eosin (HE) staining, the tissues were dissected and collected for overnight fixation with 4% PFA (pH 7.4), and further dehydrated and paraffinized with an automatic tissue processor (Leica ASP 200). The paraffin-embedded tissues were sectioned at 5 µm. Selected sections were dewaxed in Xylene followed by rehydration with a graded ethanol series (100-75-50-25%) before being stained with Harris hematoxylin and Eosin.
For whole-mount IHC, the tissues were first dissected and cultured followed by fixation and permeabilization with ice-cold 100% methanol for 10 min before the overnight incubation with primary antibodies. Next day the tissues were vigorously washed with PBST (0.1% Tween 20 in PBS) several times (5-8x 1h) after which the samples were further incubated with the relevant species-specific secondary antibodies overnight before imaging.
For immunofluorescence on sections, the tissues were dissected and collected for overnight fixation with 4% PFA (pH 7.4). The selected paraffin sections were gradually rehydrated through an ethanol series after deparaffinization followed by heat induced antigen retrieval in antigen retrieving buffer (20mM Tris, 1mM EDTA; pH 8.5). After blocking with 10% fetal bovine serum (Hyclone) for 1 hour at room temperature, the sections were incubated with primary antibody overnight at 4 ℃ followed by incubation with the relevant species-specific secondary antibodies conjugated with Alexa Fluor® 488, Alexa Fluor® 594 or Alexa Fluor® 647 respectively, and stained for nuclei with Hoechst. The information of the primary and the secondary antibodies used is shown in S1 table.

Whole mount live imaging, trunk measurements and cell counting
Urogenital blocks were dissected at E11.5 for half an hour culture after which they were stained with anti-E-Cadherin antibody and anti-Sox9 antibody. All samples were imaged with 10X objective with epiluorescence microscope (Carl Zeiss) equipped with the HXP120 LED unit (Zeiss) and a high-resolution charge-coupled device camera (AxioCam HRc, Zeiss).
The measurement of primary trunk length was carried out in cultured E11.5 and intact postnatal kidneys using Zen lite software (2012 blue edition) through tracing the primary trunk manually with hand-held computer mouse (see Fig. S5A-E). Data is expressed as the average primary trunk length ± SEM and with sample size of four for wild type control and Gdnf hyper/hyper , and eleven for wild type control and seven for Gdnf wt/ko .
For live-imaging, kidney rudiments were cultured in the humidified chamber of a Marianas 3i (3I intelligent Imaging Innovations) set-up equipped with Zeiss Axio Observer Z1 fully motorized inverted microscope and motorized xy-Stage (ASI MS-2000) for a total of 24h. Images were taken every 30min and were processed for videos with Zeiss Zen software.
EdU pulse labelling was performed by applying a final concentration of 10µM EdU to the culture medium at the beginning of the culture period. After a 30min pulse, one half of the lower urogenital block of each embryo was collected for immediate ice-cold methanol fixation. The EdU-containing culture medium was replaced with normal medium for the other half of the lower urogenital block that was cultured for an additional 8 hours without EdU.
Before fixation, the tissues were washed with PBS and EdU incorporation detection was performed with Click-iT EdU Alexa Fluor Imaging Kit (Molecular Probes/Invitrogen) according the manufacturer's instructions.
Imaging for EdU and total cell count analysis was performed with LSM 700 confocal microscope (Carl Zeiss) equipped with the HXP120C LED unit (Zeiss) and a high-resolution charge-coupled device camera (AxioCam HRc, Zeiss). The kidneys were imaged with 20 X objective and Zen software (2012 SP1; black edition; 8.1.0.484) was utilized for image processing. Quantification of the number of epithelial cells was carried out by using Imaris software (version 7.2) through counting the nuclear Hoechst signal for total cell counts, and EdU signal for Data is presented as the average number of total or Edu+ epithelial cells in the tips and trunks at given time point.
For the graphs shown in Fig. 4, cell numbers at 0h as set to 100% and change in cell numbers presented as an increase or decrease to that. The graphs shown in Fig. S5F demonstrate the percentage of EdU+ cells among the total cell numbers at given time point in control and Gdnf hyper/hyper ureteric bud tips.

Imaging
Epifluorescent imaging was performed with a Zeiss Axio Imager M2 microscope equipped with a AxioCam HRc camera and Leica DM6000 microscope equipped with a HAMAMTSU Flash 4.0 camera. The images were processed with Zen software and LAS AF software, respectively. The imaging of whole-mount tissues was performed with a SteREO Lumar.V12 stereomicroscope equipped with AxioCam MRm camera (Carl Zeiss, Germany).
Confocal imaging was performed with Zeiss LSM 700 confocal microscope (Carl Zeiss, Germany) equipped with Zen software. The number of EdU-labelled ureteric bud epithelial cells and the total number of ureteric bud epithelial cells were counted from confocal images with Imaris software (Version 8.1; Bitplane).

In vivo fertility
The breeding performance of nine F1 and six F2 Gdnf wt/hyper male mice in isogenic background was followed in normal mating conditions. Two-month-old males were mated with wt C57Bl/6N female mice, and the number of litters and offspring of each male was recorded during a time period of six months. The capability to mate was 5 tested by overnight breeding followed by examination of vaginal plug appearance in females mated with four F1 and three F2 Gdnf wt/hyper male mice in isogenic background. Values were compared to those obtained from Gdnf wt/hyper in mixed background male mice in breeding at the same time and in the same animal room. S1    Arrowhead points to the region in WT common nephric duct, which shows clear signal for apoptosis. Cleaved-