1. Author Response:

    Reviewer #1 (Public Review):

    [...] Strengths:

    1. The loss of ciliary GPR161 has a more robust phenotype in specific tissues (i.e., the limbs and face). As a result, the limb data (in Figure 6) and craniofacial data (in Figure 7) are well presented and clear. In these figures, the authors directly compare and highlight differences between primarily two genotypes (wt and Gpr161mut1/mut1 embryos) and quantify the changes (digit number and distance between nasal pits). Overall, these two figures support the existing GPR161 model, showcasing that a loss of ciliary GPR161 results in a tissue-specific loss of GLI3R (Figure 6D) and consequently the development of additional digits (Figure 6E) and craniofacial defects (Figure 7D and 7E).

    Thank you.

    Weaknesses:

    1. There is no data in the paper showing that Gli3 repressor function is affected preferentially compared to Gli Activator function. In Figure 4C, Gli3 FL/R ratios are not different between wt/wt and mut/mut embryos. The data can be explained by the fact that the mutant Gpr161 is a partial loss of function allele and the resultant weaker phenotypes (compared to the full KO) show some tissue specificity. Linking this allele to a specific biochemical mechanism is not justified by the data.

    We have now revised the title of the paper and the discussion emphasizing on these limitations. We have also added a new section in discussion on the limitations of our methods and other optogenetic/chemogenetic methods for generating cAMP in cilia. These limitations arise from the cilioplasm not being strictly restricted from the cytoplasm. Therefore, the second messengers cAMP and Ca2+ are freely diffusible between ciliary and extraciliary compartments (Delling et al., 2016; Truong et al., 2021). A paper published in Cell during revision of this study used optogenetic tools to show that ciliary, but not cytoplasmic, production of cAMP functions through PKA localized in cilia (Truong et al., 2021) to repress sonic hedgehog-mediated somite patterning in zebrafish (Wolff et al., 2003). We have also compared and discussed these results with our study. Our study highlights that the effects of ciliary loss of Gpr161 pools are tissue specific and dependent on the requirements of the tissues on GliR vs GliA in the morpho-phenotypic spectrum. Overall, our results using Gpr161mut1 allele are complementary to the optogenetic study by showing that lack of ciliary Gpr161 pools result in Hh hyperactivation phenotypes arising mainly from lack of GliR, in the limb buds, mid-face and intermediate neural tube.

    1. The authors use an endpoint assay based on overexpression in 293T cells to claim that cAMP production is unaffected by the Gpr161mut allele. However, weak effects (very likely given the weak phenotypes) may not be evident this assay. We also do not know if the mutant allele is defective in some other biochemical function or in localization to other places in the cell. One way to address this is to measure ciliary and extraciliary cAMP in their knock-in cells. In Gpr161mut1/mut1 cells, is ciliary cAMP reduced to levels comparable to Gpr161ko/ko cells? Is extraciliary cAMP unchanged compared to WT cells? Or, is cAMP able to diffuse into the cilia from GPR161mut1 localized to vesicles at the ciliary base (Figure 1B)? Many of the conclusions made in the paper equate a loss of ciliary GPR161 to a loss of ciliary cAMP, but this loss of ciliary cAMP is not definitively shown in the paper.

    As physiological ligands for Gpr161 are currently not known, we are unable to test extraciliary vs ciliary contribution of Gpr161 in cAMP production in a physiological context. Therefore, we resort to overexpression assays for constitutive cAMP production by Gpr161 and Gpr161mut1. Using these assays, we do not find a difference in constitutive activity among these variants.

    As the cilioplasm is not strictly compartmentalized from the cytoplasm, the second messengers cAMP and Ca2+ are freely diffusible between ciliary and extraciliary compartments (Delling et al., 2016; Truong et al., 2021). Thus, in any approach for generating subcellular pools of cAMP, be it genetic, optogenetic or chemogenetic (Guo et al., 2019; Hansen et al., 2020; Truong et al., 2021), extraciliary cAMP could diffuse into ciliary compartments. A recent paper using optogenetic and chemogenetic tools for cAMP production inside cilia or in cytoplasm show that there is free access of cytoplasmic cAMP to intraciliary compartments but is unable to reach critical thresholds in activating PKA (Truong et al., 2021). Thus, we would assume that the extraciliary cAMP produced by extra copies of Gpr161mut1 could diffuse to cilia but is likely to be less effective in activating downstream effectors. In addition, the PKA regulatory subunit-AKAP complexes are fundamentally important in organizing and sustaining PKA catalytic subunit activation to organize localized substrate phosphorylation in restrictive nanodomains (Bock et al., 2020; Zhang et al., 2020). The dual functions of Gpr161 in Gs coupling and as an atypical AKAP (Bachmann et al., 2016) is likely to further restrict cAMP signaling in ciliary or extraciliary microdomains.

    1. Compared to Figures 6 and 7, the data presented in Figures 3 and 5 are very confusing and difficult to interpret. On the one hand, this is understandable, the Gpr161mut/mut phenotypes are complex, and some tissues (like the developing spinal cord) are more resistant to change due to a loss of GliR. On the other hand, the data collected from the numerous genotypes analyzed could be easier to interpret by (i) providing a penetrance of the phenotypes and (ii) quantifying the phenotypes.

    Thank you for all the suggestions. We have now carried out these quantifications or tabulations, which have considerably improved the presentation of the datasets (Table 2 and Figure 5-figure supplement 1). Some of these experiments required additional experimental animals (Table 1), and we have updated the text accordingly.

    Below are a few examples of data that could be improved with quantifications:

    — In Figure 3, the authors are trying to convey that the Gpr161mut allele is partially functional and produces a milder phenotype than the Gpr161ko allele. However, the Gpr161ko/ko, Gpr161mut/ko, and Gpr161mut/mut phenotypes showcased in the figure all look quite severe, and it is difficult to appreciate the differences in the defects fully. An accompanying table summarizing the phenotypes and their penetrance in the affected genotypes would help to convey this point.

    We have added an accompanying Table 2 summarizing the phenotypes and penetrance for the respective genotypes, when present. Please note that rostral malformations such as exencephaly are similar between Gpr161 ko/ko and Gpr161 ko/mut1, whereas Gpr161 mut1/mut1 embryos have mid face widening. In the same line, Gpr161 ko/ko has no forelimbs, whereas Gpr161 ko/mut1 has smaller fore limb buds, whereas Gpr161 mut1/mut1 embryos have polydactyly.

    — In Table 1, the authors note that the Gpr161mut1/mut1 mouse is embryonic lethal by e14.5, but the analysis in Table 1 appears to be incomplete. In the table titled "breeding between Gpr161 mut1/+ parents," the authors indicate that they only assessed one litter of e14.5 and e15.5 embryos. Oddly, the authors note that additional litters were collected, but the embryos were not genotyped because the embryos exhibited no phenotypes. The absence of phenotypes could be due to an absence of viable Gpr161mut1/mut1 embryos; however, the embryos need to be genotyped and a chi-square analysis conducted to verify this. Death can be a measure of phenotype severity, but I think it is important to surmise why the embryos are dying. It is unclear whether the embryos are dying due to the heart defects mentioned in the discussion. If the embryos are dying due to the heart defect, then it would be important to know whether the heart defects are more severe in the Gpr161ko/ko embryos.

    Our apologies for the oversight. We have now analyzed additional timed pregnancies at E14.5, E14.75 and E15.5. We find that the embryonic lethality is seen fully by E14.75. Heart defects in Gpr161 ko/ko embryos are not apparent as they are E10.5 lethal. We do see apparent heart defect phenotypes in Gpr161 ko/mut1 vs Gpr161 mut1/mut1. These defects include pericardial effusion, outflow tract defects, A-V cushion abnormalities and smaller ventricles. These phenotypic descriptions are beyond the scope of the current paper. However, we have mentioned about pericardial effusion in the text and Table 2.

    — In Figure 5, quantifying the progenitor domains would greatly assist in discerning differences between the various genotypes. For example, a quantification would help readers assess differences in NKX6.1 across the various genotypes.

    We have now quantified the differences in Nkx6.1 across genotypes. The data is presented in Figure 5-figure supplement 1.

    On an unrelated note, the PAX7 staining of the Gpr161mut1/ko spinal cord looks very strange because the line adjacent to the image does not accurately represent the dorsal-ventral patterning of PAX7 seen in the image. This image would need to be replaced.

    Our apologies for the oversight. We have now revised this image.

    Reviewer #2 (Public Review):

    The premise of the entire study is predicated on GPR161mut1 failing to target to cilia and being WT in every other aspect. The Gs coupling of GPR161mut1 is examined. The ciliary localization ofGPR161mut1 is carefully assessed by conducting staining not just in WT cells but also in INPP5Ecells where GPR161 ciliary levels are known to be elevated. Another prediction is that GPR161mut1is found in an intermediate biosynthetic compartment. Some insights into the compartment whereGPR161mut1 is found would help interpret the phenotype of the GPR161mut1 animals. It would be important to know whether the GPR161mut1 mimics a pre-cilia targeted GPR161 (say at the plasma membrane) or whether it mimics a post-ciliary exit state (say recycling endosomes). In the past few years, work from the von Zastrow lab and others has shown that GPCRs keep activating their downstream partners after endocytosis from the plasma membrane. If GPR161mut1 were to mimic the post-ciliary exit state of GPR161, it may assume some of the signaling functions of ciliaryGPR161.

    Thank you for all the suggestions. We have now examined and extensively discussed the plausible source of extraciliary Gpr161 in mediating Hh repression. We already showed that Gpr161 localizes to the periciliary recycling endosomal compartment where it localizes in addition to cilia (Mukhopadhyay et al., 2013) and could activate ACs and PKA in proximity to the centrosome. We now show that Gpr161mut1 also localizes to similar compartments (Figure 1-figure supplement 3). We propose that this compartment could promote Gpr161 activity outside cilia in the in vivo settings in GliR formation (please see model in Figure 8D).

    We also compare our results with a recently published paper showing that ciliary, but not cytopasmic, production of cAMP functions through PKA localized in cilia to repress sonic hedgehog-mediated somite patterning in zebrafish (Truong et al., 2021). While this paper is an elegant demonstration of ciliary pools of cAMP in repressing Hh activity despite having no strict compartmentalization exclusively in cilia, it does not capture the roles of ciliary and extraciliary pools of Gpr161-mediated cAMP signaling in different tissues that we show are dependent on the requirements of the tissues on GliR vs GliA in the morpho-phenotypic spectrum.

    A second point that the authors may wish to address is whether GPR161mut1 may fail to enrich in cilia because it is hyperactive and undergoes constitutive exit from cilia. The hypothesis here is thatGPR161mut1 couples to beta arrestin better than WT GPR161. Blocking GPR161mut1 exit via depletion of beta arrestin or BBSome is a simple way to test this hypothesis.

    As advised by the reviewer, we have tested for Gpr161/Gpr161mut1 levels in cilia upon arrestin1/2 or BBSome loss. These experiments show that Gpr161mut1 is not present in cilia in arrestin1/2 (Arrb1/2) double ko MEFs (Figure 1-figure supplement 1) or upon RNAi of BBS4 (Figure 5-figure supplement 2). We previously also showed that knockdown of the 5’phosphpatase INPP5E that causes accumulation of Gpr161 in cilia does not show any accumulation of Gpr161mut1 in cilia. Based on all these experiments, we surmise that Gpr161mut1 does not transit through cilia.

    Finally, it would be good to learn about the levels of expression of GPR161mut1 compared to WTGPR161 using immunoblotting. If GPR161mut1 were to be expressed at much higher levels than WTGPR161, it may compensate for its lack of ciliary localization by elevated total cellular activity.

    We were unable to determine protein stability of the mutant receptor in the Gpr161mut1 embryos due to technical constraints in immunoblotting for endogenous levels. However, we note Gpr161mut1 in vesicles surrounding the base of cilia (Figure 1B) and constitutive cAMP signaling activity (Figure 1G, Figure supplements 1-3) in stable cell lines, suggesting that protein levels and activity of the mutant were comparable with wild type Gpr161. As suggested by the reviewer, we also tested LAP-tagged Gpr161mut1protein levels by tandem affinity purification and immunoblotting, with respect to LAP-tagged Gpr161wt in MEFs stably overexpressing these variants. We noted similar immunoblotting pattern from receptor glycosylation in both variants (Figure 2-figure supplement 2).

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  2. Evaluation Summary:

    This paper will be of interest to scientists interested in the biology of the G-protein-coupled receptor Gpr161 and its ciliary regulation of Hedgehog (Hh) signaling. The phenotypes observed in a new GPR161 mutant mouse carrying a hypomorphic allele provide additional information showing which developing tissues are more sensitive to Gpr161 function. However, the data at this stage are insufficient to support the main novel conclusion: the ciliary function of Gpr161 is to regulate Gli3 repressor while the extra-ciliary function is to regulate Gli activator.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    The authors created a new GPR161 mutant mouse (Gpr161mut/mut) in which GPR161 does not localize to the primary cilium but is still cAMP signaling competent based on an over-expression assay in 293T cells. Through a detailed analysis of the Gpr161mut/mut mouse and its comparison to a previously generated Gpr161 knockout mouse (Gpr161ko/ko), the authors try to discriminate the ciliary and non-ciliary roles of GPR161. The current prevailing model is that GPR161 (localized to the primary cilium in the absence of Hh pathway activation) is constitutively active and elevates cAMP levels within the primary cilium. Elevated ciliary cAMP then activates ciliary (or ciliary adjacent) PKA, driving the processing of bifunctional GLI proteins into transcriptional repressors (GLIR). According to this model, the ciliary pool of GPR161 is critical for suppressing Hh signaling activity, and one would predict that the Gpr161mut/mut embryos would look identical to the Gpr161ko/ko embryos. However, this was not the case. Across multiple developmental tissues, the Gpr161mut/mut phenotype is less severe than the complete knockout, suggesting a role for non-ciliary GPR161 in suppressing Hh signaling activity. The observations made in this paper are interesting, but the data fails to make a clear distinction between the ciliary and non-ciliary roles of GPR161.

    Strengths:

    1. The loss of ciliary GPR161 has a more robust phenotype in specific tissues (i.e., the limbs and face). As a result, the limb data (in Figure 6) and craniofacial data (in Figure 7) are well presented and clear. In these figures, the authors directly compare and highlight differences between primarily two genotypes (wt and Gpr161mut1/mut1 embryos) and quantify the changes (digit number and distance between nasal pits). Overall, these two figures support the existing GPR161 model, showcasing that a loss of ciliary GPR161 results in a tissue-specific loss of GLI3R (Figure 6D) and consequently the development of additional digits (Figure 6E) and craniofacial defects (Figure 7D and 7E).

    Weaknesses:

    1. There is no data in the paper showing that Gli3 repressor function is affected preferentially compared to Gli Activator function. In Figure 4C, Gli3 FL/R ratios are not different between wt/wt and mut/mut embryos. The data can be explained by the fact that the mutant Gpr161 is a partial loss of function allele and the resultant weaker phenotypes (compared to the full KO) show some tissue specificity. Linking this allele to a specific biochemical mechanism is not justified by the data.

    2. The authors use an endpoint assay based on overexpression in 293T cells to claim that cAMP production is unaffected by the Gpr161mut allele. However, weak effects (very likely given the weak phenotypes) may not be evident this assay. We also do not know if the mutant allele is defective in some other biochemical function or in localization to other places in the cell. One way to address this is to measure ciliary and extraciliary cAMP in their knock-in cells. In Gpr161mut1/mut1 cells, is ciliary cAMP reduced to levels comparable to Gpr161ko/ko cells? Is extraciliary cAMP unchanged compared to WT cells? Or, is cAMP able to diffuse into the cilia from GPR161mut1 localized to vesicles at the ciliary base (Figure 1B)? Many of the conclusions made in the paper equate a loss of ciliary GPR161 to a loss of ciliary cAMP, but this loss of ciliary cAMP is not definitively shown in the paper.

    3. Compared to Figures 6 and 7, the data presented in Figures 3 and 5 are very confusing and difficult to interpret. On the one hand, this is understandable, the Gpr161mut/mut phenotypes are complex, and some tissues (like the developing spinal cord) are more resistant to change due to a loss of GliR. On the other hand, the data collected from the numerous genotypes analyzed could be easier to interpret by (i) providing a penetrance of the phenotypes and (ii) quantifying the phenotypes. Below are a few examples of data that could be improved with quantifications:

    — In Figure 3, the authors are trying to convey that the Gpr161mut allele is partially functional and produces a milder phenotype than the Gpr161ko allele. However, the Gpr161ko/ko, Gpr161mut/ko, and Gpr161mut/mut phenotypes showcased in the figure all look quite severe, and it is difficult to appreciate the differences in the defects fully. An accompanying table summarizing the phenotypes and their penetrance in the affected genotypes would help to convey this point.

    — In Table 1, the authors note that the Gpr161mut1/mut1 mouse is embryonic lethal by e14.5, but the analysis in Table 1 appears to be incomplete. In the table titled "breeding between Gpr161 mut1/+ parents," the authors indicate that they only assessed one litter of e14.5 and e15.5 embryos. Oddly, the authors note that additional litters were collected, but the embryos were not genotyped because the embryos exhibited no phenotypes. The absence of phenotypes could be due to an absence of viable Gpr161mut1/mut1 embryos; however, the embryos need to be genotyped and a chi-square analysis conducted to verify this. Death can be a measure of phenotype severity, but I think it is important to surmise why the embryos are dying. It is unclear whether the embryos are dying due to the heart defects mentioned in the discussion. If the embryos are dying due to the heart defect, then it would be important to know whether the heart defects are more severe in the Gpr161ko/ko embryos.

    — In Figure 5, quantifying the progenitor domains would greatly assist in discerning differences between the various genotypes. For example, a quantification would help readers assess differences in NKX6.1 across the various genotypes. On an unrelated note, the PAX7 staining of the Gpr161mut1/ko spinal cord looks very strange because the line adjacent to the image does not accurately represent the dorsal-ventral patterning of PAX7 seen in the image. This image would need to be replaced.

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  4. Reviewer #2 (Public Review):

    The premise of the entire study is predicated on GPR161mut1 failing to target to cilia and being WT in every other aspect. The Gs coupling of GPR161mut1 is examined. The ciliary localization of GPR161mut1 is carefully assessed by conducting staining not just in WT cells but also in INPP5E cells where GPR161 ciliary levels are known to be elevated. Another prediction is that GPR161mut1 is found in an intermediate biosynthetic compartment. Some insights into the compartment where GPR161mut1 is found would help interpret the phenotype of the GPR161mut1 animals. It would be important to know whether the GPR161mut1 mimics a pre-cilia targeted GPR161 (say at the plasma membrane) or whether it mimics a post-ciliary exit state (say recycling endosomes). In the past few years, work from the von Zastrow lab and others has shown that GPCRs keep activating their downstream partners after endocytosis from the plasma membrane. If GPR161mut1 were to mimic the post-ciliary exit state of GPR161, it may assume some of the signaling functions of ciliary GPR161.

    A second point that the authors may wish to address is whether GPR161mut1 may fail to enrich in cilia because it is hyperactive and undergoes constitutive exit from cilia. The hypothesis here is that GPR161mut1 couples to beta arrestin better than WT GPR161. Blocking GPR161mut1 exit via depletion of beta arrestin or BBSome is a simple way to test this hypothesis.

    Finally, it would be good to learn about the levels of expression of GPR161mut1 compared to WT GPR161 using immunoblotting. If GPR161mut1 were to be expressed at much higher levels than WT GPR161, it may compensate for its lack of ciliary localization by elevated total cellular activity.

    Read the original source
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