GPR17 is an Essential Component of the Negative Feedback Loop of the Sonic Hedgehog Signalling Pathway in Neural Tube Development

Dorsal-ventral pattern formation of the neural tube is regulated by temporal and spatial activities of extracellular signalling molecules. Sonic hedgehog (Shh) assigns ventral neural subtypes via activation of the Gli transcription factors. Shh activity changes dynamically during neural differentiation, but the mechanisms responsible for regulating this dynamicity are not fully understood. Here we show that the P2Y-type G-protein coupled receptor GPR17 is involved in temporal regulation of the Shh signal. GPR17 was expressed in the ventral progenitor regions of the neural tube and acted as a negative regulator of the Shh signal in chick embryos. While the activation of the GPR17-related signal inhibited ventral identity, perturbation of GPR17 expression led to aberrant expansion of ventral neural domains. Notably, perturbation of GPR17 expression partially inhibited the negative feedback of Gli activity. Moreover, GPR17 increased cAMP activity, suggesting that it exerts its function by inhibiting the processing of Gli3 protein. GPR17 also negatively regulated Shh signalling in neural cells differentiated from mouse embryonic stem cells, suggesting that GPR17 function is conserved among different organisms. Our results demonstrate that GPR17 is a novel negative regulator of Shh signalling in a wide range of cellular contexts. Author Summary During neural development, determination of cell fate and the progress of differentiation are regulated by extracellular signal molecules, including Sonic Hedgehog (Shh). Shh forms a gradient within the embryonic organ of the central nervous system, or the neural tube, and a variety of cells are produced corresponding to the concentration. While the signal concentration is critical for cell fate, recent studies have revealed that the intracellular signal intensity does not always correspond to the Shh concentration. Rather, the intracellular signal intensity changes over time. Importantly, the signal intensity peaks and gradually decreases thereafter, and the half-life of the Shh signal contributes to the cell fate determination. However, the mechanisms for this temporal change are not fully understood. By using chick embryos and mouse embryonic stem cells as model systems, we demonstrate that the G-protein coupled receptor, GPR17, is an essential regulator for the negative feedback of the Shh signal during neural development. While GPR17 gene expression is induced by the Shh signal, GPR17 perturbs the Shh signalling pathway. This negative function of GPR17 on the Shh signal is conserved among different vertebrate species. The collective data demonstrate that GPR17 is a negative regulator for the Shh signalling pathway in a wide range of the cellular contexts.


5 8
This effect occurred independently from the programmed cell death, as the increasing number of  To confirm that Shh activity was decreased by GPR17-mediated signalling, we performed a 2 6 1 reporter assay to measure Gli activity. We prepared pools of explants transfected with the GBS-Luc 2 6 2 reporter construct, which harboured the luciferase gene driven by the Gli binding sequence (GBS).

6 3
The luciferase activity was measured after 24 hours. As the result, Gli activity was significantly 2 6 4 upregulated by Shh H (Fig 3L lanes 1 and 2). However, when MDL29951 was added along with Shh H , 2 6 5 the activity was reduced to a level close to that of explants cultured with the Shh L (more than four 2 6 6 pools of explants in each condition; Fig 3L lanes 2-4). These data confirmed that the Gli activity was 2 6 7 perturbed by GPR17 and its related signals.

6 8
We attempted to further investigate if the activation of the GPR17-related signal correlated 2 6 9 with the elevation of the intracellular cAMP level, by assaying intermediate neural explants.

7 0
Consistent with the previous observation, the cAMP level in the explants treated with Shh for 24 hours 2 7 1 showed a lower cAMP level than in the control explants ( Fig 3M, lanes 1 and 2)   in the cAMP level.

7 5
Together, these findings suggested that the GPR17-mediated signalling pathway negatively 2 7 6 regulates Shh activity in the context of neural tube pattern formation through the upregulation of the 2 7 7 intracellular cAMP level.  intracellular Shh signalling, and is essential for proper pattern formation in the neural tube. Given that expression of GPR17 is induced by Shh, and GPR17 negatively regulates the Shh 3 0 3 signal activity at the intracellular level ( Fig 1B, 1D, 2E, 3E-3L), we reasoned that GPR17 is involved 3 0 4 in temporal regulation of Gli activity [13][14][15]. To test this hypothesis, we analysed the role of GPR17 3 0 5 in temporal regulation of the Gli activity.

0 6
First, to confirm that the intracellular Shh signal activity was aberrantly upregulated, we

1 6
We next sought to analyse dynamic control of the intracellular Shh signal activity during 3 1 7 ventral neural differentiation. For this purpose, we performed luciferase reporter assays using GBS-

1 8
Luc at various time points. We prepared explants electroporated with GBS-Luc with either si-control 3 1 9 or si-GPR17, and then measured Gli activity at a series of time points from 6 to 48 hours after the 3 2 0 initiation of the culture. In si-control-electroporated explants treated with Shh L , Shh activity gradually 3 2 1 decreased over time, peaking at 6 hours and becoming undetectable by the 48 hour time point (more 3 2 2 than 3 explants per point; Fig 5J, blue line), consistent with previous reports [13,14]. On the other 3 2 3 hand, in explants with si-GPR17, Gli activity at 6 hours was comparable to that of the si-control 3 2 4 explants, but the activity was significantly higher at 24 hours and still detectable at 36 hours ( Fig 5J, 3 2 5 red line).

2 6
We next attempted to confirm that the dynamic Gli activity reflects the gene expression. As

4
This result supports the idea that the dynamic Gli activity was affected, at least partially, by the 3 3 5 perturbation of the GPR17 expression.

6
In addition, we calculated the ratio of Gli3FL and Gli3R in the neural tube. For this purpose, 3 3 7 we prepared isolated neural tubes that had been electroporated with either si-control or si-GPR17, and 3 3 8 analysed the forms of Gli3 by western blotting. We found that the ratio of Gli3FL over Gli3R was

4 1
Together, these findings indicate that GPR17 is an essential upstream factor that controls the 3 4 2 dynamic change between the full-length and repressor forms of Gli3, and thus regulates the temporal 3 4 3 change in the intracellular Shh signalling activity.

0 5
Although GPR17 has been suggested to be a receptor for the uracil nucleotides and Cysteinyl

1 1
In this study, we utilised MDL29951 to experimentally activate GPR17 (Fig 3F, 3H [27], GPR17 expression is firstly recognised at e14.5 in the mouse brain using reporter gene 4 2 3 expression. The difference between our data and the previous findings is presumably caused by the 4 2 4 protein stability of GPR17 or the difference in the detection methods.

2 5
While our analysis revealed the essential roles of GPR17 on the dorsal-ventral pattern 4 2 6 formation of the spinal cord in chick, the effect in the mouse neural tube has been unknown, and at 4 2 7 least, does not seem to be critical [27,45,46], as GPR17-deficient mice are viable. This is probably due 4 2 8 to the redundant roles of the multiple GPCRs expressed in the neural tube. The expression of Adenylyl 4 2 9 cyclase 5 (AC5), which catalases the dissociation of ATP to make cAMP [20,21], was found to be suggesting that it has diverse and cell type-specific functions.

0
Concerning the relationships between GPCR and Shh signal, two GPCRs, GPR161 and

6 2
Although Shh induces the GPR17 expression (Fig 1D), GPR17 does not seem to be a direct

8 6
This noise in the signal is constantly modulated by negative feedback, and consequently the reliability 4 8 7 and reproducibility of pattern formation can be incarnated [57,58]. Therefore, the negative feedback  GPCRs that can couple with Gα q or Gα s were identified by referring to the website 4 9 7 (http://gpcrdb.org), and qPCR primers were designed against the corresponding genes (S1 Table).

9 8
Relative expression levels were analysed by RT-qPCR in neural explants treated or untreated with 4 9 9 Shh H (S1 Fig). NCBI Gene IDs for chicken and mouse GPR17 are 769024 and 574402, respectively. protein VP16, as described previously [55]. Embryos were fixed with 4% paraformaldehyde,

1 4
Cryosections were cut at 14 μm increments and analysed with immunohistochemistry or in situ 5 1 5 hybridisation. The sections from at least five independent embryos were analysed, and the number of

8 9
For the cAMP assay in the NIH3T3 cells (Fig 2H), the cells were transfected with the 5 9 0 indicated plasmids and the transfected cells were selected for 24 hours. 3-isobutyl-1-methylxanthine 5 9 1 (IBMX) was added at 1 μM in the last 30 minutes before cells were harvested. For the cAMP assay in 5 9 2 the neural explants (Fig 3I), 15 explants were prepared for each condition and cultured for 24 hours.

9 3
IBMX was added in the last 1 hour. The cAMP assay was performed using DetectX high sensitivity