Zebrafish GPR161 Contributes to Basal Hedgehog Repression in a Tissue-specific Manner

Hedgehog (Hh) ligands act as morphogens to direct patterning and proliferation during embryonic development. Protein kinase A (PKA) is a central negative regulator of Hh signalling, and in the absence of Hh ligands, PKA activity prevents inappropriate expression of Hh target genes. The Gαs- coupled receptor Gpr161 contributes to the basal Hh repression machinery by activating PKA, although the extent of this contribution is unclear. Here we show that loss of Gpr161 in zebrafish leads to constitutive activation of low-, but not high-level Hh target gene expression in the neural tube. In contrast, in the myotome, both high- and low-level Hh signalling is constitutively activated in the absence of Gpr161 function. Our results suggest that the relative contribution of Gpr161 to basal repression of Hh signalling is tissue-specific. Distinct combinations of G-protein-coupled receptors may allow the fine-tuning of PKA activity to ensure the appropriate sensitivity to Hh across different tissues.


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The Hh signalling pathway is a key regulator of cell fate specification and proliferation during 27 embryonic development, and plays important roles in adult tissue homeostasis (Briscoe and Thérond, 28 2013;Ingham et al., 2011). Dysregulation of Hh signalling can lead to the formation of common and 29 severe forms of human cancers such as basal cell carcinoma and medulloblastoma (Jiang and Hui, 30 2008;Raleigh and Reiter, 2019). 31 When Hh ligands bind their receptor Patched (Ptch), the inhibition of Smo by Ptch is alleviated, and 32 Smo localises to the primary cilium (Corbit et al., 2005), where it activates downstream signalling to 33 regulate the activity of the bifunctional Gli transcription factors. 34 Hh ligands act as morphogens, and the transcriptional outcome of Hh signalling is determined by the 35 balance between repressor and activator forms of the Gli transcription factors. This balance is 36 controlled by the activity of PKA and other kinases (Hui and Angers, 2011;Niewiadomski et al., 2019). 37 In the absence of Hh, the basal Hh repression machinery is thought to maintain a high level of PKA 38 activity. PKA phosphorylates the Gli proteins and primes them for further phosphorylation and 39 proteolytic cleavage, to yield truncated forms that act as transcriptional repressors (GliR) 40 (Niewiadomski et al., 2014;Pan et al., 2009;Wang et al., 2000). In addition, PKA also plays a role in 41 restricting the activity of full length Gli (GliA) by promoting its association with Sufu (Humke et al., 42 2010;Marks and Kalderon, 2011). Low levels of exposure to Hh ligands blocks the formation of GliR,43 whereas high levels of Hh exposure is required for the formation of the activator forms of Gli. This is 44 thought to be controlled through a cluster of six PKA target residues in Gli, with distinct 45 phosphorylation patterns regulating the formation of repressor and activator forms (Niewiadomski et 46 al., 2014). This rheostat mechanism ensures that the level of Gli transcriptional activity corresponds 47 ptch2 -/double mutant embryos (Koudijs et al., 2008). In contrast to ptch1 -/-; ptch2 -/double mutant 125 embryos which lack eyes (Koudijs et al., 2008), a rudimentary eye can be identified in gpr161 126 mutants at 72 hpf ( Figure 2B). 127 At this stage, the retina of wild type embryos is organised into six evolutionarily conserved layers: the 128 pigmented epithelium, the photoreceptor cell layer, the outer plexiform layer, the inner nuclear 129 layer, the inner plexiform layer, and the ganglion cell layer (Schmitt and Dowling, 1999). Semi-thin 130 sectioning revealed that while eye morphogenesis was abnormal, remnants of all six layers were 131 clearly identified by morphology ( Figure 2D commonly seen in mutants of negative regulators of Hh signalling, such as sufu, ptch1 and hhip 146 (Whitfield et al., 1996), and suggests that Gpr161 acts to negatively regulate Hh signalling also in 147 zebrafish. 148 Gpr161 mutant mice do not form limb buds (Hwang et al., 2018;Mukhopadhyay et al., 2013) and 149 ptch1 -/-; ptch2 -/double mutant zebrafish embryos lack pectoral fin buds (Koudijs et al., 2008). In Hedgehog signalling is upregulated in gpr161 mutant embryos

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The morphological phenotypes observed in gpr161 mutants are consistent with increased Hh 154 signalling. To determine whether Hh signalling is upregulated in gpr161 mutants, we assessed the 155 expression of known Hh target genes in the neural tube by qRT-PCR and RNA in situ hybridisation. 156 The Hh target genes ptch2, gli1, nkx2.2b and nkx6.1 ( Figure 3B) were all strongly upregulated, while pax7a, which is repressed by Hh signalling (Guner and Karlstrom, 2007), was strongly downregulated 158 in gpr161 mutant compared to wild type embryos ( Figure 3B). RNA in situ hybridisation revealed that 159 expression of shha, the major Hh ligand expressed in the neural plate was not expanded in gpr161 160 mutants ( Figure 3A). However, expression of ptch2, a direct transcriptional target of Hh signalling 161 (Concordet et al., 1996), was expanded in gpr161 mutants ( Figure 3A), suggesting that Hh signalling is 162 upregulated in gpr161 mutants downstream of Shh expression. Similarly, expression of olig2, a 163 marker of motor neuron induction which depends on low-level Hh activity (Dessaud et al., 2007;Park 164 et al., 2002), as well as nkx2.2a, a marker for V3 interneuron progenitor cells of the lateral floorplate 165 (Barth and Wilson, 1995;Briscoe et al., 1999), were clearly expanded in the gpr161 mutant neural 166 tube ( Figure 3A). We note that the expansion of the low-level target olig2 appears to be stronger 167 than the expansion of the high-level target nkx2.2a ( Figure 3A). Taken together, these results show 168 that loss of Gpr161 leads to a hyperactivation of the Hh signalling pathway in zebrafish. 169 In the zebrafish myotome, sustained Hh signalling during gastrulation and somitogenesis stages have 170 been shown to be required for the specification of several cell types, including Prox1 and Eng double 171 positive muscle pioneer cells (MPs) and Prox1 positive superficial slow fibres (SSFs) (Wolff et al., 172 2003). While medium-to-low level Hh signalling is sufficient for the specification of SSFs, the 173 formation of MPs requires high levels of Hh (Wolff et al., 2003). Consistent with the expansion of Hh 174 target genes in the neural tube, gpr161 mutants also displayed an increase in both SSFs and MPs 175 ( Figure 3C). While zygotic Gpr161 loss of function resulted in a significant increase in both SSFs (from 176 22 ± 2 (mean ±SD) in wt to 33 ±5 in zygotic gpr161 mutants) and MPs (from 4 ± 1 (mean ± SD) in wt 177 to 7 ± 2 in zygotic gpr161 mutants), complete loss of both maternal and zygotic Gpr161 led to a 178 stronger increase in both SSFs and MPs (56 ± 9 (mean ± s.d.) SSFs, and 23 ± 10 MPs), consistent with 179 the requirement for sustained Hh signalling in muscle cell development (Wolff et al., 2003). These 180 results suggest that in the somites, loss of Gpr161 results in expansion of both high and low Hh 181 signalling targets. 182 gpr161 mutants remain sensitive to Shh

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Our results suggest that although loss of Gpr161 function in zebrafish leads to increased Hh signalling 184 activity, gpr161 mutants display weaker phenotypes than those seen in mutants with maximal 185 activation of Hh signalling. To determine whether Hh signalling could be further activated in gpr161 186 mutants in response to Hh, we injected 50 or 100 pg shh mRNA, and assessed SSF and MP formation 187 using Prox1 and Eng antibody staining as above ( Figure 4A A comparison of phenotypes suggests that the upregulation of Hh signalling in zebrafish gpr161 259 mutants is less severe than that seen in ptc1 -/-; ptc2 -/mutant embryos (Koudijs et al., 2005). Similarly, 260 injection of a dominant-negative form of PKA resulted in an apparently stronger increase in Hh-261 dependent muscle cell specification in the myotome than could observed in the gpr161 mutants 262 (Zhao et al., 2016). This is consistent with data obtained in mice, where a loss of PKA or Gs leads to 263 more severe ventralisation of the neural tube than what is observed in the Gpr161 mutants 264 (Mukhopadhyay et al., 2013;Pusapati et al., 2018;Regard et al., 2013;Tuson et al., 2011). Further 265 supporting the idea that Hh signalling is not maximally activated in the gpr161 mutants, we find that 266 injection of shha mRNA can further increase high, but not low, level Hh targets in the somites of 267 gpr161 mutants. Thus we conclude that whereas low level Hh signalling is maximally active in the 268 gpr161 mutants, additional mechanisms contribute to PKA activation to control high level Hh 269 signalling in the absence of Gpr161 function. can not rule out that some low level nkx2.2a expression persists in the zebrafish triple mutants. 286 Another possibility is that there could be species-specific differences in the roles of GliR and GliA 287 and/or cAMP levels, or alternatively, Gpr161 may make a relatively larger contribution to cAMP 288 levels in the zebrafish neural tube compared to mouse. Interestingly, we do observe tissue-specific 289 differences in zebrafish in our epistasis experiments. In the gpr161 mutant myotome, both high and 290 low level Hh signalling outcomes are independent of Smo, suggesting that in the somites Gpr161 is 291 completely epistatic to Smo. We suggest that in the neural tube, additional unknown factors make 292 significant contributions to promote basal cAMP levels, whereas in the zebrafish myotome, Gpr161 293 alone may account for the largest part of the basal repression machinery. Thus, distinct combinations 294 of GPCRs in different cell types can contribute to complex and tissue-specific regulation of Hh 295 signalling. The identification of these additional GPCRs, as well as other factors that control PKA 296 activity downstream of adenylate cyclases, will be required to understand how Hh signalling is fine-297 tuned to orchestrate the great variety of Hh-dependent biological processes in a cell type specific 298 manner. 299

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To generate expression-vectors for gpr161a and gpr161b the coding sequence of both genes were 319 amplified with overlapping primers (see Table 2

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RNA was isolated from zebrafish embryos with Trizol (Ambion) following the manufacturer's 324 instructions. RNA integrity was checked by agarose gel electrophoresis and the concentration was 325 measured using a Nanodrop 2000c (Thermo) spectrophotometer. 326 Complementary DNA was transcribed from equal amounts of dsDNase-treated total RNA using the 327 Maxima RT kit for qPCR (Thermo) with dsDNase according to the manufacturer's instructions. 328 RT-qPCRs were performed using 5x HOT FIREPol EvaGreen qPCR Supermix (Solis Biodyne) and 329 contained each primer at 250nM and cDNA corresponding to a total RNA amount of 15 ng for pooled 330 embryos or 5 ng for single embryos. PCRs were run on a CFX96 Connect (BioRad) under following 331 conditions: 12 min 95°C, 40 cycles of 95°C for 30s, 60°C for 30s and 72°C for 20s. Melt curves were 332 recorded from 65°C to 95°C in 0.5°C increments. Data was acquired using CFX Manager 3.1 (BioRad) 333 and exported as RDML files for processing.  Electron microscopy 372 Embryos were fixed with 2.5% glutaraldehyde in 0.01M sodium cacodylate buffer containing 5% 373 sucrose at 4°C for two hours. After washing in cacodylate buffer, specimens were post fixed in 374 reduced osmium (2% osmium tetroxide and 3% potassium ferrocyanide in 0.1M cacodylate buffer) 375 for two hours at 4°C, dehydrated in an ethanol series, critical point dried with an EMS 850 CPD 376 (Electron Microscopy Services, Germany), mounted and 20 nm gold sputtered with a CCU-010 377 sputter coater (Safematic, Switzerland), and examined with a DSM950 scanning electron microscope 378 (Zeiss, Germany). Images were taken with a Pentax digital camera and PK_Tether 0.7.0 free software. 379

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All data presented in this study were analyzed with R using the RStudio integrated development 381 environment and plotted using the "ggplot2" package (Rstudio Team, 2016;Wickham, 2016). 382 Statistical significance of differences in expression levels between groups were calculated on at least 383 three biological replicates with the Wilcoxon rank sum test, corrected for multiple comparisons using 384 the Benjamini-Hochberg FDR method.
Statistical significance of a difference in MP or SSF numbers between groups was determined using 386 one-way ANOVA corrected for unequal variances and the Games-Howell post-hoc test for pairwise 387 comparison as implemented in the "userfriendlyscience" package (Peters, 2017). P values are 388 indicated as follows: * p<0.05; ** p<0.01; p<0.001; ns not significant. Sample sizes (N)