GRK2 Kinases in the Primary Cilium Initiate SMOOTHENED-PKA Signaling in the Hedgehog Cascade.

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO, and the ensuing PKA-C binding and inactivation, are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

Ciliary ARL13B inhibits developmental kidney cystogenesis in mouse.

ARL13B is a small GTPase enriched in cilia. Deletion of Arl13b in mouse kidney results in renal cysts and an associated absence of primary cilia. Similarly, ablation of cilia leads to kidney cysts. To investigate whether ARL13B functions from within cilia to direct kidney development, we examined kidneys of mice expressing an engineered cilia-excluded ARL13B variant, ARL13BV358A. These mice retained renal cilia and developed cystic kidneys. Because ARL13B functions as a guanine nucleotide exchange factor (GEF) for ARL3, we examined kidneys of mice expressing an ARL13B variant that lacks ARL3 GEF activity, ARL13BR79Q. We found normal kidney development with no evidence of cysts in these mice. Taken together, our results show that ARL13B functions within cilia to inhibit renal cystogenesis during mouse development, and that this function does not depend on its role as a GEF for ARL3.

Ciliary ARL13B inhibits developmental kidney cystogenesis in mouse.

ARL13B is a small GTPase enriched in cilia. Deletion of Arl13b in mouse kidney results in renal cysts and an associated absence of primary cilia. Similarly, ablation of cilia leads to kidney cysts. To investigate whether ARL13B functions from within cilia to direct kidney development, we examined kidneys of mice expressing an engineered cilia-excluded ARL13B variant, ARL13BV358A. These mice retained renal cilia and developed cystic kidneys. Because ARL13B functions as a guanine nucleotide exchange factor (GEF) for ARL3, we examined kidneys of mice expressing an ARL13B variant that lacks ARL3 GEF activity, ARL13BR79Q. We found normal kidney development with no evidence of cysts in these mice. Taken together, our results show that ARL13B functions within cilia to inhibit renal cystogenesis during mouse development, and that this function does not depend on its role as a GEF for ARL3.

Smoothened and ARL13B are critical in mouse for superior cerebellar peduncle targeting.

Patients with the ciliopathy Joubert syndrome present with physical anomalies, intellectual disability, and a hindbrain malformation described as the "molar tooth sign" due to its appearance on an MRI. This radiological abnormality results from a combination of hypoplasia of the cerebellar vermis and inappropriate targeting of the white matter tracts of the superior cerebellar peduncles. ARL13B is a cilia-enriched regulatory GTPase established to regulate cell fate, cell proliferation, and axon guidance through vertebrate Hedgehog signaling. In patients, mutations in ARL13B cause Joubert syndrome. To understand the etiology of the molar tooth sign, we used mouse models to investigate the role of ARL13B during cerebellar development. We found that ARL13B regulates superior cerebellar peduncle targeting and these fiber tracts require Hedgehog signaling for proper guidance. However, in mouse, the Joubert-causing R79Q mutation in ARL13B does not disrupt Hedgehog signaling nor does it impact tract targeting. We found a small cerebellar vermis in mice lacking ARL13B function but no cerebellar vermis hypoplasia in mice expressing the Joubert-causing R79Q mutation. In addition, mice expressing a cilia-excluded variant of ARL13B that transduces Hedgehog normally showed normal tract targeting and vermis width. Taken together, our data indicate that ARL13B is critical for the control of cerebellar vermis width as well as superior cerebellar peduncle axon guidance, likely via Hedgehog signaling. Thus, our work highlights the complexity of ARL13B in molar tooth sign etiology.

The ciliary phosphatidylinositol phosphatase Inpp5e plays positive and negative regulatory roles in Shh signaling.

Sonic hedgehog (Shh) signal transduction specifies ventral cell fates in the neural tube and is mediated by the Gli transcription factors that play both activator (GliA) and repressor (GliR) roles. Cilia are essential for Shh signal transduction and the ciliary phosphatidylinositol phosphatase Inpp5e is linked to Shh regulation. In the course of a forward genetic screen for recessive mouse mutants, we identified a functional null allele of inositol polyphosphate-5-phosphatase E (Inpp5e), ridge top (rdg), with expanded ventral neural cell fates at E10.5. By E12.5, Inpp5erdg/rdg embryos displayed normal neural patterning and this correction over time required Gli3, the predominant repressor in neural patterning. Inpp5erdg function largely depended on the presence of cilia and on smoothened, the obligate transducer of Shh signaling, indicating that Inpp5e functions within the cilium to regulate the pathway. These data indicate that Inpp5e plays a more complicated role in Shh signaling than previously appreciated. We propose that Inpp5e attenuates Shh signaling in the neural tube through regulation of the relative timing of GliA and GliR production, which is important in understanding how the duration of Shh signaling regulates neural tube patterning.

Disruption of the ciliary GTPase Arl13b suppresses Sonic hedgehog overactivation and inhibits medulloblastoma formation.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor, and overactivation of the Sonic Hedgehog (Shh) signaling pathway, which requires the primary cilium, causes 30% of MBs. Current treatments have known negative side effects or resistance mechanisms, so new treatments are necessary. Shh signaling mutations, like those that remove Patched1 (Ptch1) or activate Smoothened (Smo), cause tumors dependent on the presence of cilia. Genetic ablation of cilia prevents these tumors by removing Gli activator, but cilia are a poor therapeutic target since they support many biological processes. A more appropriate strategy would be to identify a protein that functionally disentangles Gli activation and ciliogenesis. Our mechanistic understanding of the ciliary GTPase Arl13b predicts that it could be such a target. Arl13b mutants retain short cilia, and loss of Arl13b results in ligand-independent, constitutive, low-level pathway activation but prevents maximal signaling without disrupting Gli repressor. Here, we show that deletion of Arl13b reduced Shh signaling levels in the presence of oncogenic SmoA1, suggesting Arl13b acts downstream of known tumor resistance mechanisms. Knockdown of ARL13B in human MB cell lines and in primary mouse MB cell culture decreased proliferation. Importantly, loss of Arl13b in a Ptch1-deleted mouse model of MB inhibited tumor formation. Postnatal depletion of Arl13b does not lead to any overt phenotypes in the epidermis, liver, or cerebellum. Thus, our in vivo and in vitro studies demonstrate that disruption of Arl13b inhibits cilia-dependent oncogenic Shh overactivation.

A novel homozygous ARL13B variant in patients with Joubert syndrome impairs its guanine nucleotide-exchange factor activity.

ARL13B encodes for the ADP-ribosylation factor-like 13B GTPase, which is required for normal cilia structure and Sonic hedgehog (Shh) signaling. Disruptions in cilia structure or function lead to a class of human disorders called ciliopathies. Joubert syndrome is characterized by a wide spectrum of symptoms, including a variable degree of intellectual disability, ataxia, and ocular abnormalities. Here we report a novel homozygous missense variant c.[223G>A] (p.(Gly75Arg) in the ARL13B gene, which was identified by whole-exome sequencing of a trio from a consanguineous family with multiple-affected individuals suffering from intellectual disability, ataxia, ocular defects, and epilepsy. The same variant was also identified in a second family. We saw a striking difference in the severity of ataxia between affected male and female individuals in both families. Both ARL13B and ARL13B-c.[223G>A] (p.(Gly75Arg) expression rescued the cilia length and Shh defects displayed by Arl13b hennin (null) cells, indicating that the variant did not disrupt either ARL13B function. In contrast, ARL13B-c.[223G>A] (p.(Gly75Arg) displayed a marked loss of ARL3 guanine nucleotide-exchange factor activity, with retention of its GTPase activities, highlighting the correlation between its loss of function as an ARL3 guanine nucleotide-exchange factor and Joubert syndrome.

Mutation of murine Sox4 untranslated regions results in partially penetrant perinatal lethality.

BACKGROUND: Sox4 is an essential gene, and genetic deletion results in embryonic lethality. In an effort to develop mice with tissue-specific deletion, we bred conditional knockout mice bearing LoxP recombination sites flanking the Sox4 gene, with the LoxP sites located in the Sox4 5'UTR and 3'UTR. RESULTS: The number of mice homozygous for this LoxP-flanked conditional knockout allele was far below the expected number, suggesting embryonic lethality with reduced penetrance. From over 200 animals bred, only 11% were homozygous Sox4(flox/flox) mice, compared to the expected Mendelian ratio of 25% (p<0.001). Moreover, there was a significant reduction in the number of female Sox4(flox/flox) mice (26%) relative to male Sox4(flox/flox) mice (p=0.0371). Reduced Sox4 expression in homozygous embryos was confirmed by in-situ hybridization and Quantitative real-time polymerase chain reaction (QPCR). CONCLUSION: LoxP sites in the 5' and 3' UTR of both alleles of Sox4 resulted in reduced, but variable expression of Sox4 message.

Multiplex Chromosomal Exome Sequencing Accelerates Identification of ENU-Induced Mutations in the Mouse.

Forward genetic screens in Mus musculus have proved powerfully informative by revealing unsuspected mechanisms governing basic biological processes. This approach uses potent chemical mutagens, such as N-ethyl-N-nitrosourea (ENU), to randomly induce mutations in mice, which are then bred and phenotypically screened to identify lines that disrupt a specific biological process of interest. Although identifying a mutation using the rich resources of mouse genetics is straightforward, it is unfortunately neither fast nor cheap. Here we show that detecting newly induced causal variants in a forward genetic screen can be accelerated dramatically using a methodology that combines multiplex chromosome-specific exome capture, next-generation sequencing, rapid mapping, sequence annotation, and variation filtering. The key innovation of our method is multiplex capture and sequence that allows the simultaneous survey of both mutant, parental, and background strains in a single experiment. By comparing variants identified in mutant offspring with those found in dbSNP, the unmutagenized background strains, and parental lines, induced causative mutations can be distinguished immediately from preexisting variation or experimental artifact. Here we demonstrate this approach to find the causative mutations induced in four novel ENU lines identified from a recent ENU screen. In all four cases, after applying our method, we found six or fewer putative mutations (and sometimes only a single one). Determining the causative variant was then easily achieved through standard segregation approaches. We have developed this process into a community resource that will speed up individual labs' ability to identify the genetic lesion in mutant mouse lines; all of our reagents and software tools are open source and available to the broader scientific community.

RIP3 mediates the embryonic lethality of caspase-8-deficient mice.

Apoptosis and necroptosis are complementary pathways controlled by common signalling adaptors, kinases and proteases; among these, caspase-8 (Casp8) is critical for death receptor-induced apoptosis. This caspase has also been implicated in non-apoptotic pathways that regulate Fas-associated via death domain (FADD)-dependent signalling and other less defined biological processes as diverse as innate immune signalling and myeloid or lymphoid differentiation patterns. Casp8 suppresses RIP3-RIP1 (also known as RIPK3-RIPK1) kinase complex-dependent necroptosis that follows death receptor activation as well as a RIP3-dependent, RIP1-independent necrotic pathway that has emerged as a host defence mechanism against murine cytomegalovirus. Disruption of Casp8 expression leads to embryonic lethality in mice between embryonic days 10.5 and 11.5 (ref. 7). Thus, Casp8 may naturally hold alternative RIP3-dependent death pathways in check in addition to promoting apoptosis. We find that RIP3 is responsible for the mid-gestational death of Casp8-deficient embryos. Remarkably, Casp8(-/-)Rip3(-/-) double mutant mice are viable and mature into fertile adults with a full immune complement of myeloid and lymphoid cell types. These mice seem immunocompetent but develop lymphadenopathy by four months of age marked by accumulation of abnormal T cells in the periphery, a phenotype reminiscent of mice with Fas-deficiency (lpr/lpr; also known as Fas). Thus, Casp8 contributes to homeostatic control in the adult immune system; however, RIP3 and Casp8 are together completely dispensable for mammalian development.

Conserved residues of the bare lymphocyte syndrome transcription factor RFXAP determine coordinate MHC class II expression.

RFXAP is required for the transcriptional regulation of MHC-II genes. Mutations in RFXAP are the genetic basis for complementation group D cases of the bare lymphocyte syndrome (BLS) immunodeficiency. Comparative genomic sequence analysis was conducted and found that only the C-terminal half of the protein is conserved among vertebrates. The C-terminal third of RFXAP, which contained an extensive glutamine-rich tract, could rescue HLA-DR, but not HLA-DQ or HLA-DP expression in a BLS cell line. To understand this phenomenon, a detailed analysis of the role of specific sequences in the C-terminal third of RFXAP with respect to MHC-II regulation was undertaken. Surprisingly, mutation of the conserved glutamine residues had no effect on activity, whereas mutation of hydrophobic and other conserved residues resulted in discoordinate MHC-II isotype expression. Moreover, mutation of potential phosphorylation sites abolished RFXAP activity. The ability of RFXAP mutants to rescue one isotype, but not another was investigated by their ability to form RFX complexes, bind DNA in vivo, recruit CIITA to promoters and to activate a series of chimeric reporter genes. The results suggest that certain RFXAP mutants exaggerate isotype promoter-specific differences and form transcriptionally inefficient activation complexes with factors at the neighboring cis-acting elements. These results show a distinction in factor recognition that is associated with specific MHC-II isotypes and may explain the basis of allele-specific expression differences.

A hierarchy of nuclear localization signals governs the import of the regulatory factor X complex subunits and MHC class II expression.

Comprised of RFX5, RFXAP, and RFX-B/ANK, the regulatory factor X (RFX) complex is an obligate transcription factor required for the expression of MHC class II genes. RFX functions by binding to the conserved X1 box sequence located upstream of all MHC class II genes. Using a mutagenesis scheme and a yeast heterologous reporter system, the mechanism by which the RFX complex is transported into the nucleus was examined. The results have identified specific nuclear localization signals (NLS) in both RFX5 and RFXAP that direct the nuclear translocation and expression of MHC class II genes. Additionally, a nuclear export signal was identified in the N terminus of RFXAP. RFX-B was poorly localized to the nucleus, and no specific NLS was identified. Whereas RFX5 could import an RFXAP NLS mutant into the nucleus, it had no effect on the import of RFX-B. The results suggest that although RFX5 and RFXAP could assemble before nuclear import, RFX-B association with the complex does not take place until after the subunits enter the nucleus. The identification of nuclear import and export sites on RFX molecules provides potential targets to modulate MHC class II expression.