Ciliary signaling in stem cells in health and disease: Hedgehog pathway and beyond.

The primary cilium is a hair-like sensory compartment that protrudes from the cellular surface. The primary cilium is enriched in a variety of signaling molecules that regulate cellular activities. Stem cells have primary cilia. They reside in a specialized environment, called the stem cell niche. This niche contains a variety of secreted factors, and some of their receptors are localized in the primary cilia of stem cells. Here, we summarize the current understanding of the function of cilia in compartmentalized signaling in stem cells. We describe how ciliary signaling regulates stem cells and progenitor cells during development, tissue homeostasis and tumorigenesis. We summarize our understanding of cilia regulated signaling -primary involving the hedgehog pathway- in stem cells in diverse settings that include neuroepithelial cells, radial glia, cerebellar granule neuron precursors, hematopoietic stem cells, hair follicle stem cells, bone marrow mesenchymal stem cells and mammary gland stem cells. Overall, our review highlights a variety of roles that ciliary signaling plays in regulating stem cells throughout life.

Feedback control of the Gpr161-Gαs-PKA axis contributes to basal Hedgehog repression in zebrafish.

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 orphan G-protein-coupled receptor Gpr161 contributes to the basal Hh repression machinery by activating PKA. Gpr161 acts as an A-kinase-anchoring protein, and is itself phosphorylated by PKA, but the functional significance of PKA phosphorylation of Gpr161 in the context of Hh signalling remains unknown. Here, we show that loss of Gpr161 in zebrafish leads to constitutive activation of medium and low, but not maximal, levels of Hh target gene expression. Furthermore, we find that PKA phosphorylation-deficient forms of Gpr161, which we show directly couple to Gαs, display an increased sensitivity to Shh, resulting in excess high-level Hh signalling. Our results suggest that PKA feedback-mediated phosphorylation of Gpr161 may provide a mechanism for fine-tuning Gpr161 ciliary localisation and PKA activity.

Enterotoxigenic Escherichia coli infection promotes enteric defensin expression via FOXO6-METTL3-m6A-GPR161 signalling axis.

The production of natural antimicrobial peptides has emerged as an important mechanism of innate immunity in animals. Defensins, members of a large family of antimicrobial peptides, have been suggested as effector molecules in host defence against bacteria, fungi, protozoa and enveloped viruses. However, the molecular mechanism underlying defensin upregulation in bacterial infection remains poorly understood. The modification of mRNA by N6-adenosine methylation (m6A) on internal bases influences gene expression in eukaryotes. Here, we show that ß-defensin production triggered by Enterotoxigenic Escherichia coli K88 (E. coli K88) infection is controlled by the cellular m6A methyltransferase METTL3. Adding back with METTL3 robustly stimulated the re-expression of defensin, which further supports the conclusion. Furthermore, using a MeRIP-seq approach, we identified a functional connection between m6A dependent GPR161 signalling and the expression of defensins. Mechanistically, we found that the transcription factor FOXO6 interacted with METTL3 to trigger the transcription of GPR161 and the subsequent regulation of ß-defensin expression. The study has shed light on the mechanisms by which enterotoxigenic Escherichia coli infection promotes enteric defensin expression.

Derepression of sonic hedgehog signaling upon Gpr161 deletion unravels forebrain and ventricular abnormalities.

Inverse gradients of transcriptional repressors antagonize the transcriptional effector response to morphogens. However, the role of such inverse regulation might not manifest solely from lack of repressors. Sonic hedgehog (Shh) patterns the forebrain by being expressed ventrally; however, absence of antagonizing Gli3 repressor paradoxically cause insufficient pathway activation. Interestingly, lack of the primary cilia-localized G-protein-coupled receptor, Gpr161 increases Shh signaling in the mouse neural tube from coordinated lack of Gli3 repressor and Smoothened-independent activation. Here, by deleting Gpr161 in mouse neuroepithelial cells and radial glia at early mid-gestation we detected derepression of Shh signaling throughout forebrain, allowing determination of the pathophysiological consequences. Accumulation of cerebrospinal fluid (hydrocephalus) was apparent by birth, although usual causative defects in multiciliated ependymal cells or aqueduct were not seen. Rather, the ventricular surface was expanded (ventriculomegaly) during embryogenesis from radial glial overproliferation. Cortical phenotypes included polymicrogyria in the medial cingulate cortex, increased proliferation of intermediate progenitors and basal radial glia, and altered neocortical cytoarchitectonic structure with increased upper layer and decreased deep layer neurons. Finally, periventricular nodular heterotopia resulted from disrupted neuronal migration, while the radial glial scaffold was unaffected. Overall, suppression of Shh pathway during early mid-gestation prevents ventricular overgrowth, and regulates cortical gyration and neocortical/periventricular cytoarchitecture.

Hedgehog receptor function during craniofacial development.

The Hedgehog signalling pathway plays a fundamental role in orchestrating normal craniofacial development in vertebrates. In particular, Sonic hedgehog (Shh) is produced in three key domains during the early formation of the head; neuroectoderm of the ventral forebrain, facial ectoderm and the pharyngeal endoderm; with signal transduction evident in both ectodermal and mesenchymal tissue compartments. Shh signalling from the prechordal plate and ventral midline of the diencephalon is required for appropriate division of the eyefield and forebrain, with mutation in a number of pathway components associated with Holoprosencephaly, a clinically heterogeneous developmental defect characterized by a failure of the early forebrain vesicle to divide into distinct halves. In addition, signalling from the pharyngeal endoderm and facial ectoderm plays an essential role during development of the face, influencing cranial neural crest cells that migrate into the early facial processes. In recent years, the complexity of Shh signalling has been highlighted by the identification of multiple novel proteins that are involved in regulating both the release and reception of this protein. Here, we review the contributions of Shh signalling during early craniofacial development, focusing on Hedgehog receptor function and describing the consequences of disruption for inherited anomalies of this region in both mouse models and human populations.

The orphan GPCR, Gpr161, regulates the retinoic acid and canonical Wnt pathways during neurulation.

The vacuolated lens (vl) mouse mutation arose on the C3H/HeSnJ background and results in lethality, neural tube defects (NTDs) and cataracts. The vl phenotypes are due to a deletion/frameshift mutation in the orphan GPCR, Gpr161. A recent study using a null allele demonstrated that Gpr161 functions in primary cilia and represses the Shh pathway. We show the hypomorphic Gpr161(vl) allele does not severely affect the Shh pathway. To identify additional pathways regulated by Gpr161 during neurulation, we took advantage of naturally occurring genetic variation in the mouse. Previously Gpr161(vl-C3H) was crossed to different inbred backgrounds including MOLF/EiJ and the Gpr161(vl) mutant phenotypes were rescued. Five modifiers were mapped (Modvl: Modifier of vl) including Modvl5(MOLF). In this study we demonstrate the Modvl5(MOLF) congenic rescues the Gpr161(vl)-associated lethality and NTDs but not cataracts. Bioinformatics determined the transcription factor, Cdx1, is the only annotated gene within the Modvl5 95% CI co-expressed with Gpr161 during neurulation and not expressed in the eye. Using Cdx1 as an entry point, we identified the retinoid acid (RA) and canonical Wnt pathways as downstream targets of Gpr161. QRT-PCR, ISH and IHC determined that expression of RA and Wnt genes are down-regulated in Gpr161(vl/vl) but rescued by the Modvl5(MOLF) congenic during neurulation. Intraperitoneal RA injection restores expression of canonical Wnt markers and rescues Gpr161(vl/vl) NTDs. These results establish the RA and canonical Wnt as pathways downstream of Gpr161 during neurulation, and suggest that Modvl5(MOLF) bypasses the Gpr161(vl) mutation by restoring the activity of these pathways.

The novel linkage between Fuz and Gpr161 genes regulates sonic hedgehog signaling during mouse embryonic development.

Sonic hedgehog (Shh) signaling regulates embryonic morphogenesis utilizing primary cilia, the cell antenna acting as a signaling hub. Fuz, an effector of planar cell polarity (PCP) signaling, involves Shh signaling via cilia formation, while the G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling. The range of phenotypic malformations observed in mice bearing mutations in either of these two genes is similar; however, their functional relations have not been previously explored. This study identified the genetic and biochemical link between Fuz and Gpr161 in mouse embryonic development. Fuz was genetically epistatic to Gpr161 via Shh signaling during mouse embryonic development. The FUZ biochemically interacted with GPR161, and Fuz regulated Gpr161 ciliary trafficking via ß-arrestin2. Our study suggested the novel Gpr161-Fuz axis that regulates Shh signaling during mouse embryonic development.

In vivo and in vitro anti-inflammation of Rhapontici Radix extract on mastitis via TMEM59 and GPR161.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhapontici Radix ethanol extract (RRE) is derived from the dried root of Rhaponticum uniflorum (L.) DC belonging to the Asteraceae family. RRE exhibits significant anti-inflammatory and antioxidant properties; however, the potential of RRE in mastitis treatment requires further investigation. AIM OF THIS STUDY: This research was performed to examine the protective properties of RRE against mastitis and the mechanisms underlying the effects of RRE. MATERIAL AND METHODS: RRE components were analyzed by HPLC-MS/MS and DPPH methods. Isochlorogenic acid B (ICAB) was obtained commercially. MTT assay was utilized to assess RRE or ICAB cytotoxicity in bovine mammary alveolar (MAC-T) cells. Immunohistochemistry were used to investigate the pathological alterations in mammary tissue. The protein levels of inflammatory cytokines and mediators were analyzed using ELISA, and the expression of MAPK and NF-κB signaling pathways, as well as p65 nuclear translocation, were analyzed through Western blotting and immunofluorescence techniques, respectively. Target proteins of RRE were screened by RNA-seq and tandem mass tag analyses. Protein interaction was revealed and confirmed using co-immunoprecipitation and CRISPR/Cas9-based knockdown and overexpression of target genes. RESULTS: ICAB was revealed as one of the main components in RRE, and it was responsible for 84.33% of RRE radical scavenging activity. Both RRE and ICAB mitigated the infiltration of T lymphocytes in the mammary glands of mice, leading to decreased levels of inflammatory mediators (COX-2 and iNOS) and cytokines (TNF-α, IL-6, and IL-1ß) in lipopolysaccharide (LPS)-induced MAC-T cells. Furthermore, RRE and ICAB suppressed the LPS-induced phosphorylation of NF-κB inhibitor and p65, thereby impeding p65 nuclear translocation in mouse mammary glands and MAC-T cells. In addition, RRE and ICAB attenuated the LPS-triggered activation of c-Jun N-terminal kinase 1/2, p38, and extracellular regulated protein kinase 1/2. Importantly, co-treated with LPS and ICAB in MAC-T cells, an upregulation of G-protein coupled receptor 161 (GPR161) and transmembrane protein 59 (TMEM59) was observed; the interact between TMEM59 and was found, leading to inhibition of NF-κB activity and inflammatory cytokine production. CONCLUSION: ICAB is a prominent antioxidant in RRE. RRE and ICAB reduce mammary inflammation via MAPK and NF-κB pathways and the interaction between TMEM59 and GPR161 mediates the control of ICAB in NF-κB signaling.

Perspectives on the implications of carrying putative pathogenic variants in the medulloblastoma predisposition genes ELP1 and GPR161.

Recent genetic sequencing studies in large series' of predominantly childhood medulloblastoma have implicated loss-of-function, predominantly truncating, variants in the ELP1 and GPR161 genes in causation of the MBSHH subtype specifically. The latter association, along with a report of an index case with some features of Gorlin syndrome has led to speculation that GPR161 may also cause Gorlin syndrome. We show that these genes are associated with relatively low absolute risks of medulloblastoma from extrapolating lifetime risks in the general population and odds ratios from the population database gnomAD. The projected risks are around 1 in 270-430 for ELP1 and 1 in 1600-2500 for GPR161. These risks do not suggest the need for MRI screening in infants with ELP1 or GPR161 variants as this is not currently recommended for PTCH1 where the risks are equivalent or higher. We also screened 27 PTCH1/SUFU pathogenic variant-negative patients with Gorlin syndrome for GPR161 and found no suspicious variants. Given the population frequencies of 0.0962% for GPR161 and 0.0687% for ELP1, neither of these genes can be a cause of Gorlin syndrome with an unexplained population frequency far lower at 0.0021%.

Rgl-exomiR-7972, a novel plant exosomal microRNA derived from fresh Rehmanniae Radix, ameliorated lipopolysaccharide-induced acute lung injury and gut dysbiosis.

Plant-derived exosome-like nanoparticles (ELNs) have been proposed as a novel therapeutic tool for preventing human diseases. However, the number of well-verified plant ELNs remains limited. In this study, the microRNAs in ELNs derived from fresh Rehmanniae Radix, a well-known traditional Chinese herb for treating inflammatory and metabolic diseases, were determined by using microRNA sequencing to investigate the active components in the ELNs and the protection against lipopolysaccharide (LPS)-induced acute lung inflammation in vivo and in vitro. The results showed that rgl-miR-7972 (miR-7972) was the main ingredient in ELNs. It exerted stronger protective activities against LPS-induced acute lung inflammation than catalpol and acteoside, which are two well-known chemical markers in this herb. Moreover, miR-7972 decreased the production of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α), reactive oxygen species (ROS) and nitric oxide (NO) in LPS-exposed RAW264.7 cells, thereby facilitating M2 macrophage polarization. Mechanically, miR-7972 downregulated the expression of G protein-coupled receptor 161 (GPR161), activating the Hedgehog pathway, and inhibited the biofilm form of Escherichia coli via targeting virulence gene sxt2. Therefore, miR-7972 derived from fresh R. Radix alleviated LPS-induced lung inflammation by targeting the GPR161-mediated Hedgehog pathway, recovering gut microbiota dysbiosis. It also provided a new direction for gaining novel bioactivity nucleic acid drugs and broadening the knowledge on cross-kingdom physiological regulation through miRNAs.

Pax3 lineage-specific deletion of Gpr161 is associated with spinal neural tube and craniofacial malformations during embryonic development.

Sonic hedgehog (Shh) signaling is the morphogen signaling that regulates embryonic craniofacial and neural tube development. G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling, and its inactivation in mice results in embryo lethality associated with craniofacial defects and neural tube defects. However, the structural defects of later embryonic stages and cell lineages underlying abnormalities have not been well characterized due to the limited lifespan of Gpr161 null mice. We found that embryos with Pax3 lineage-specific deletion of Gpr161 presented with tectal hypertrophy (anterior dorsal neuroepithelium), cranial vault and facial bone hypoplasia (cranial neural crest), vertebral abnormalities (somite) and the closed form of spina bifida (posterior dorsal neuroepithelium). In particular, the closed form of spina bifida was partly due to reduced Pax3 and Cdx4 gene expression in the posterior dorsal neural tubes of Gpr161 mutant embryos with decreased Wnt signaling, whereas Shh signaling was increased. We describe a previously unreported role for Gpr161 in the development of posterior neural tubes and confirm its role in cranial neural crest- and somite-derived skeletogenesis and midbrain morphogenesis in mice.

Primary cilia, A-kinase anchoring proteins and constitutive activity at the orphan G protein-coupled receptor GPR161: A tale about a tail.

Primary cilia are non-motile antennae-like structures responsible for sensing environmental changes in most mammalian cells. Ciliary signalling is largely mediated by the Sonic Hedgehog (Shh) pathway, which acts as a master regulator of ciliary protein transit and is essential for normal embryonic development. One particularly important player in primary cilia is the orphan G protein-coupled receptor, GPR161. In this review, we introduce GPR161 in the context of Shh signalling and describe the unique features on its C-terminus such as PKA phosphorylation sites and an A-kinase anchoring protein motif, which may influence the function of the receptor, cAMP compartmentalisation and/or trafficking within primary cilia. We discuss the recent putative pairing of GPR161 and spexin-1, highlighting the additional steps needed before GPR161 could be considered 'deorphanised'. Finally, we speculate that the marked constitutive activity and unconventional regulation of GPR161 may indicate that the receptor may not require an endogenous ligand.

Ciliary signaling proteins are mislocalized in the brains of Bardet-Biedl syndrome 1-null mice.

In the brain, primary cilia are found on most, if not all, central neurons. The importance of neuronal cilia is underscored by the fact that human diseases caused by primary cilia dysfunction, which are known as ciliopathies, are associated with neuropathologies, including neuropsychiatric disorders and learning and memory deficits. Neuronal cilia are enriched for certain G protein-coupled receptors and their downstream effectors, suggesting they sense and respond to neuromodulators in the extracellular milieu. GPCR ciliary localization is disrupted in neurons from mouse models of the ciliopathy Bardet-Biedl syndrome, with GPCRs failing to localize to cilia, indicating the Bardet-Biedl syndrome proteins are required for trafficking of G protein-coupled receptors into neuronal cilia. Yet, dopamine receptor 1 accumulates in cilia in the absence of Bardet-Biedl syndrome proteins, suggesting Bardet-Biedl syndrome proteins are required for normal ciliary import and export. To further explore the roles of the Bardet-Biedl syndrome proteins in neuronal cilia, we examined localization of ciliary signaling proteins in a new constitutive Bbs1 knockout mouse model. Interestingly, we find that two additional ciliary G protein-coupled receptors (Gpr161 and Gpr19) abnormally accumulate in cilia on Bardet-Biedl syndrome neurons. In addition, we find that the GPCR signaling protein ß-arrestin accumulates in a subset of cilia in the brain, suggesting the presence of additional unidentified ciliary G protein-coupled receptors. These results confirm the importance of the Bardet-Biedl syndrome proteins in establishing ciliary GPCR pathways and indicate that loss of Bbs1 leads to complex changes in the localization of signaling proteins in the brain.

Wnt1 Lineage Specific Deletion of Gpr161 Results in Embryonic Midbrain Malformation and Failure of Craniofacial Skeletal Development.

Sonic hedgehog (Shh) signaling regulates multiple morphogenetic processes during embryonic neurogenesis and craniofacial skeletal development. Gpr161 is a known negative regulator of Shh signaling. Nullizygous Gpr161 mice are embryonic lethal, presenting with structural defects involving the neural tube and the craniofacies. However, the lineage specific role of Gpr161 in later embryonic development has not been thoroughly investigated. We studied the Wnt1-Cre lineage specific role of Gpr161 during mouse embryonic development. We observed three major gross morphological phenotypes in Gpr161 cKO (Gpr161 f/f; Wnt1-Cre) fetuses; protrusive tectum defect, encephalocele, and craniofacial skeletal defect. The overall midbrain tissues were expanded and cell proliferation in ventricular zones of midbrain was increased in Gpr161 cKO fetuses, suggesting that protrusive tectal defects in Gpr161 cKO are secondary to the increased proliferation of midbrain neural progenitor cells. Shh signaling activity as well as upstream Wnt signaling activity were increased in midbrain tissues of Gpr161 cKO fetuses. RNA sequencing further suggested that genes in the Shh, Wnt, Fgf and Notch signaling pathways were differentially regulated in the midbrain of Gpr161 cKO fetuses. Finally, we determined that cranial neural crest derived craniofacial bone formation was significantly inhibited in Gpr161 cKO fetuses, which partly explains the development of encephalocele. Our results suggest that Gpr161 plays a distinct role in midbrain development and in the formation of the craniofacial skeleton during mouse embryogenesis.

Ciliary and extraciliary Gpr161 pools repress hedgehog signaling in a tissue-specific manner.

The role of compartmentalized signaling in primary cilia during tissue morphogenesis is not well understood. The cilia localized G protein-coupled receptor, Gpr161, represses hedgehog pathway via cAMP signaling. We engineered a knock-in at the Gpr161 locus in mice to generate a variant (Gpr161mut1), which was ciliary localization defective but cAMP signaling competent. Tissue phenotypes from hedgehog signaling depend on downstream bifunctional Gli transcriptional factors functioning as activators or repressors. Compared to knockout (ko), Gpr161mut1/ko had delayed embryonic lethality, moderately increased hedgehog targets, and partially down-regulated Gli3 repressor. Unlike ko, the Gpr161mut1/ko neural tube did not show Gli2 activator-dependent expansion of ventral-most progenitors. Instead, the intermediate neural tube showed progenitor expansion that depends on loss of Gli3 repressor. Increased extraciliary receptor levels in Gpr161mut1/mut1 prevented ventralization. Morphogenesis in limb buds and midface requires Gli repressor; these tissues in Gpr161mut1/mut1 manifested hedgehog hyperactivation phenotypes-polydactyly and midfacial widening. Thus, ciliary and extraciliary Gpr161 pools likely establish tissue-specific Gli repressor thresholds in determining morpho-phenotypic outcomes.

Identification and spatiotemporal expression of gpr161 genes in zebrafish.

G protein coupled Receptor 161 (GPR161) is a ciliary orphan GPCR. It is reported to play critical roles in regulating vertebrate Hedgehog (Hh) signaling pathway, that is conserved in metazoan and functions in earlier embryogenesis and homeostasis of adult metabolism. However, to date, all GPR161 functional studies were performed only in mouse. Knock out gpr161 in NIH3T3 cell lines, the common material for Hh mechanism research, failed to give any obvious Hh pathway defects, raising the question that whether GPR161 functions in Hh pathway is conserved in vertebrate system. Here, we described the characterization and spatiotemporal expression of two zebrafish gpr161 homologs, gpr161a and gpr161b. gpr161a was renamed of the gpr161 previously identified, while gpr161b was novel identified. The whole-mount in situ hybridization and quantitative PCR results showed that gpr161a is initially expressed in maternal manner while gpr161b is not. Although these two gpr161 showed ubiquitously expressed at early embryonic stages, each of them had tissue specific accumulation. gpr161a is abundant in the central nervous system (CNS) and adaxial cells, where are rich of Hh responding cells. Together gpr161a was highly expressed in muscle and intestine in adult fishes. These results strongly suggest the regulating roles of Gpr161 a in zebrafish Hh signal transduction. gpr161b was also accumulated in the CNS but mainly at the midline in the neural tube, similar pattern as wnt5b expression in such area, suggesting its potential function correlated with WNT signaling pathway. Interestingly, we also found the specific accumulation of gpr161 in posterior blood island (PBI) at 24 hours post fertilization (hpf), indicating the gpr161 may play roles in early hematopoiesis in zebrafish. Our work provides a starting point to unveil the divergent functions of gpr161 in vertebrate and will shed light on the studies of mechanism of Hh and WNT pathways, as well as early hematopoiesis.

Requirement of IFT-B-BBSome complex interaction in export of GPR161 from cilia.

The intraflagellar transport (IFT) machinery, which includes the IFT-A and IFT-B complexes, mediates bidirectional trafficking of ciliary proteins. In addition to these complexes, the BBSome, which is composed of eight subunits that are encoded by the causative genes of Bardet-Biedl syndrome (BBS), has been proposed to connect the IFT machinery to ciliary membrane proteins, such as G protein-coupled receptors, to mediate their export from cilia. However, little is known about the connection between the IFT machinery and the BBSome. Using the visible immunoprecipitation assay, we here identified the interaction between IFT38 from the IFT-B complex and BBS1, BBS2 and BBS9 from the BBSome. Furthermore, by analyzing phenotypes of IFT38-knockout cells exogenously expressing wild-type IFT38 or its mutant lacking the ability to interact with BBS1+BBS2+BBS9, we showed that knockout cells expressing the IFT38 mutant have restored ciliogenesis; however, similar to BBS1-knockout cells, they demonstrated significant accumulation of GPR161 within cilia upon stimulation of Hedgehog signaling. These results indicate that the IFT-B-BBSome interaction is required for the export of GPR161 across the ciliary gate.

Basal Suppression of the Sonic Hedgehog Pathway by the G-Protein-Coupled Receptor Gpr161 Restricts Medulloblastoma Pathogenesis.

Sonic hedgehog (Shh) determines cerebellar granule cell (GC) progenitor proliferation and medulloblastoma pathogenesis. However, the pathways regulating GC progenitors during embryogenesis before Shh production by Purkinje neurons and their roles in tumorigenesis remain unclear. The cilium-localized G-protein-coupled receptor Gpr161 suppresses Shh-mediated signaling in the neural tube. Here, by deleting Gpr161 in mouse neural stem cells or GC progenitors, we establish Gpr161 as a tumor suppressor in Shh subtype medulloblastoma. Irrespective of Shh production in the cerebellum, Gpr161 deletion increased downstream activity of the Shh pathway by restricting Gli3-mediated repression, causing more extensive generation and proliferation of GC progenitors. Moreover, earlier deletion of Gpr161 during embryogenesis increased tumor incidence and severity. GC progenitor overproduction during embryogenesis from Gpr161 deletion was cilium dependent, unlike normal development. Low GPR161 expression correlated with poor survival of SHH subtype medulloblastoma patients. Gpr161 restricts GC progenitor production by preventing premature and Shh-dependent pathway activity, highlighting the importance of basal pathway suppression in tumorigenesis.

G-protein-coupled receptor signaling and neural tube closure defects.

Disruption of the normal mechanisms that mediate neural tube closure can result in neural tube defects (NTDs) with devastating consequences in affected patients. With the advent of next-generation sequencing, we are increasingly detecting mutations in multiple genes in NTD cases. However, our ability to determine which of these genes contribute to the malformation is limited by our understanding of the pathways controlling neural tube closure. G-protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors in humans and have been historically favored as drug targets. Recent studies implicate several GPCRs and downstream signaling pathways in neural tube development and closure. In this review, we will discuss our current understanding of GPCR signaling pathways in pathogenesis of NTDs. Notable examples include the orphan primary cilia-localized GPCR, Gpr161 that regulates the basal suppression machinery of sonic hedgehog pathway by means of activation of cAMP-protein kinase A signaling in the neural tube, and protease-activated receptors that are activated by a local network of membrane-tethered proteases during neural tube closure involving the surface ectoderm. Understanding the role of these GPCR-regulated pathways in neural tube development and closure is essential toward identification of underlying genetic causes to prevent NTDs. Birth Defects Research 109:129-139, 2017. © 2016 Wiley Periodicals, Inc.