Tamara Caspary

Primary cilia coordinate signaling pathways that regulate tissue homeostasis and development, and defects in cilia contribute to numerous ciliopathies. However, the transcriptional consequences of disrupting ciliary protein localization remain poorly defined. ARL13B is a cilia-enriched regulatory GTPase required for ciliary trafficking and signaling. The ARL13BV358A variant is undetectable in cilia yet retains known biochemical functions, providing a unique model to investigate the functions of ciliary ARL13B independently of ciliogenesis. To define transcriptional programs associated with loss of ciliary ARL13B, we generated 2 independent Arl13bV358A/V358A kidney epithelial cell lines and matched rescue lines. The ARL13BV358A mutation did not affect ciliation frequency or cilia length but altered ciliary protein composition, including loss of ARL3 and INPP5E localization and increased accumulation of TULP3. RNA sequencing revealed expression changes in genes associated with ciliary biology, mechanotransduction, epithelial organization, and kidney-related phenotypes. Despite similar ciliary phenotypes, the independently derived mutant clones displayed substantial transcriptomic heterogeneity, highlighting a potential source of variation in CRISPR-based transcriptional studies. By integrating data from the independent mutant and rescue clones, we identified a high-confidence set of 131 genes whose expression reproducibly tracked with loss and restoration of ciliary ARL13B. Together, these findings demonstrate that ciliary ARL13B is required to maintain normal ciliary composition and gene expression programs and underscore the value of multiclone, rescue-based experimental designs for robust transcriptomic analyses.

Key Points: Loss of ciliary ARL13B suppressed cystogenesis in the kidneys of an adult mouse model of polycystic kidney disease without ablating cilia. Loss of ciliary ARL13B or the residue critical for its guanine nucleotide exchange factor activity did not affect kidney morphology or physiology in an adult induced mouse model. Mutation of a residue critical for ARL13B activation of ARL3 suppressed cystic phenotypes in Pkd1 -dependent cysts. Background: Polycystic kidney disease (PKD) is the leading genetic cause of kidney failure, resulting in the accumulation of fluid-filled cysts and gross enlargement of the kidney. Mutations in PKD1 or PKD2 , which encode ciliary polycystin proteins, are the most common cause of PKD. These proteins function in a cilia-dependent cyst activation (CDCA) pathway—one that requires cilia for its procystic function—yet the molecular driver(s) of this pathway are unknown. ARL13B is a regulatory GTPase enriched in cilia and links to cystogenesis in the kidneys. ARL13B possesses guanine nucleotide exchange factor (GEF) activity for ARL3, another ADP-ribosylation factor–like family GTPase with links to cilia. Methods: We used 2 distinct Arl13b mouse alleles to investigate whether ARL13B is a component of the CDCA pathway: Arl13b ^V358A encodes for enzymatically normal ARL13B that is undetectable in cilia, and Arl13b ^R79Q encodes for cilia-localized ARL13B lacking a residue critical for its ARL3 GEF activity. We used these alleles in a Pkd1 -deficient adult mouse model and investigated kidney morphology (hematoxylin and eosin and cystic index analysis), physiology (BUN measurements), kidney fibrosis (picrosirius staining and α -smooth muscle actin levels), kidney injury (SRY box transcription factor 9 immuno-fluorescent staining and quantification), and Wnt signaling ( β -catenin and cyclin D1 protein levels). Results: We found that loss of ciliary ARL13B or mutation of a single residue critical for its ARL3 GEF activity suppressed Pkd1 -dependent cysts. We observed reductions in kidney size, cystic index, and BUN. We also observed suppression of kidney fibrosis, kidney injury, and β -catenin and cyclin D1 protein levels. Conclusions: Our results identified a subcellular location and mechanism driving Pkd1 -dependent cystogenesis in the kidneys. We demonstrated that expression of a critical residue for ARL13B's GEF activity specifically in cilia is a key mechanism of the CDCA pathway driving cystogenesis in the kidneys.

Primary cilia coordinate signaling pathways that regulate tissue homeostasis and development, and defects in cilia contribute to numerous ciliopathies. However, the transcriptional consequences of disrupting ciliary protein localization remain poorly defined. ARL13B is a cilia-enriched regulatory GTPase required for ciliary trafficking and signaling. The ARL13B ^V358A variant is undetectable in cilia yet retains known biochemical functions, providing a unique model to investigate the functions of ciliary ARL13B independently of ciliogenesis. To define transcriptional programs associated with loss of ciliary ARL13B, we generated two independent Arl13b ^V358A/V358A kidney epithelial cell lines and matched rescue lines. The ARL13B ^V358A mutation did not affect ciliation frequency or cilia length but altered ciliary protein composition, including loss of ARL3 and INPP5E localization and increased accumulation of TULP3. RNA sequencing revealed expression changes in genes associated with ciliary biology, mechanotransduction, epithelial organization, and kidney-related phenotypes. Despite similar ciliary phenotypes, the independently-derived, mutant clones displayed substantial transcriptomic heterogeneity, highlighting a potential source of variation in CRISPR-based transcriptional studies. By integrating data from the independent mutant and rescue clones, we identified a high-confidence set of 131 genes whose expression reproducibly tracked with loss and restoration of ciliary ARL13B. Together, these findings demonstrate that ciliary ARL13B is required to maintain normal ciliary composition and gene expression programs and underscores the value of multi-clone, rescue-based experimental designs for robust transcriptomic analyses. Summary for Reviewers: This study examined how excluding the protein ARL13B from primary cilia affects kidney epithelial cells. The researchers created two independent cell lines carrying a modified form of ARL13B, along with matched rescue cell lines. The findings show that ciliary ARL13B helps maintain normal ciliary composition. By comparing the cell lines, the researchers identified a high-confidence set of genes associated with loss of ciliary ARL13B. By highlighting the importance of using independent gene-edited clones and rescue-based controls, these results advance understanding of how cilia regulate kidney cell function and provide guidance for designing robust transcriptomic analyses.

Primary cilia are sensory cellular appendages that regulate diverse developmental and homeostatic processes, including energy homeostasis. In animal models and humans, their dysfunction can lead to hyperphagia and obesity. ARL13B is a regulatory GTPase enriched in cilia. We engineered an Arl13b mouse allele, Arl13bV358A, that disrupts ARL13B from localizing to primary cilia. Homozygous Arl13bV358A/V358A mice become hyperphagic, obese, and insulin-resistant. Restoring wild-type ARL13B to cilia in 4-week-old Arl13bV358A/V358A mice fully rescued the obesity and metabolic dysfunction. Additionally, the selective exclusion of ARL13B from cilia in the nervous system caused obesity. Together, these findings establish that ciliary ARL13B function within the nervous system is necessary for body weight regulation. Our ability to genetically uncouple the ciliary and nonciliary functions of ARL13B in a cell type–specific manner enables us to define its cilia-specific role and offers new insights into the molecular mechanisms underlying primary cilia control of energy homeostasis. Article Highlights: The molecular mechanisms by which primary cilia regulate energy homeostasis remain poorly understood. Here, we evaluated whether the ciliary GTPase ARL13B regulates energy homeostasis and whether its localization to cilia is required to control body weight and feeding. Using genetic tools to isolate cilia-specific functions, we found that systemic exclusion of ARL13B from cilia causes hyperphagia and obesity in mice and that ciliary ARL13B is required in the nervous system for weight control. These findings identify ciliary ARL13B as a key regulator of energy homeostasis, contributing to our understanding of how primary cilia act as metabolic signaling hubs.

Background: Polycystic kidney disease (PKD) is the leading genetic cause of renal failure, resulting in the accumulation of fluid filled cysts and gross enlargement of the kidney. Mutations in PKD1 or PKD2 , which encode ciliary polycystin proteins, are the most common cause of PKD. These proteins function in a cilia-dependent cyst activation (CDCA) pathway–one that requires cilia for its pro-cystic function–yet the molecular driver(s) of this pathway are unknown. ARL13B is a regulatory GTPase enriched in cilia and links to renal cystogenesis. ARL13B possesses guanine nucleotide exchange factor (GEF) activity for ARL3, another ARL with links to cilia. Methods: We used two distinct Arl13b mouse alleles to investigate whether ARL13B is a component of the CDCA pathway: Arl13b ^V358A encodes for enzymatically normal ARL13B that is undetectable in cilia, and Arl13b ^R79Q encodes for cilia-localized ARL13B lacking a residue critical for its ARL3 GEF activity. We used these alleles in a Pkd1 -deficient adult mouse model and investigated renal morphology (H&E and cystic index analysis), physiology (blood urea nitrogen measurements), renal fibrosis (picrosirius staining and α-smooth muscle actin levels), renal injury (SOX9 immunofluorescent staining and quantification), and Wnt signaling (β-catenin and cyclin D1 protein levels). Results: We found that loss of ciliary ARL13B or mutation of a single residue critical for its ARL3 GEF activity suppressed Pkd1 -dependent cysts. We observed reductions in kidney size, cystic index, and blood urea nitrogen. We also observed suppression of renal fibrosis, renal injury, and β-catenin and cyclin D1 protein levels. Conclusions: Our results identified a subcellular location and mechanism driving Pkd1 -dependent renal cystogenesis. We demonstrated that expression of a critical residue for ARL13B’s GEF activity specifically in cilia is a key mechanism of the CDCA pathway driving renal cystogenesis. Key Points: Loss of ciliary ARL13B suppressed renal cystogenesis in an adult mouse model of polycystic kidney disease (PKD) without ablating cilia Loss of ciliary ARL13B or mutation of the residue critical for its GEF activity did not affect renal morphology or physiology in an adult induced mouse model Mutation of a residue critical for ARL13B activation of ARL3 suppressed cystic phenotypes in Pkd1 -dependent cysts

ARL13B is a regulatory GTPase enriched in cilia, making it a popular marker for this organelle. Arl13bhnn/hnn mice lack ARL13B expression, die during mid-gestation, and exhibit defects in ciliogenesis. The R26Arl13b-Fucci2aR biosensor mouse line directs the expression of fluorescently tagged full-length Arl13b cDNA upon Cre recombination. To determine whether constitutive, ubiquitous expression of Cerulean-tagged ARL13B (ARL13B-Cerulean) can replace endogenous gene expression, we generated Arl13bhnn/hnn animals expressing ARL13B-Cerulean. We show that Arl13bhnn/hnn;Arl13b-Cerulean mice survive to adulthood with no obvious physical or behavioral defects, indicating that the fluorescently tagged protein can functionally replace the endogenous protein during development. However, we observed that rescued males failed to sire offspring, revealing a role for ARL13B in spermatogenesis. This work shows that the R26Arl13b-Fucci2aR mouse contains an inducible allele of Arl13b capable of functioning in most tissues and biological processes.

Astrocyte cilia are largely understudied due to the lack of available tools. Astrocyte research advanced with the establishment of Aldh1l1-CreERT2, an inducible Cre line that specifically targets the astrocyte lineage. Here, we develop and compare genetic models that label astrocyte cilia in the developing prefrontal cortex (PFC) using Aldh1l1-CreERT2 and Cre-dependent cilia reporters. We evaluate these models by testing different tamoxifen-induction protocols and quantifying the percentage of astrocytes labeled with the cilia reporters. We show that tamoxifen dosage impacts the expression of cilia reporters in astrocytes. We uncover the maximum cilia-labeling efficiency using recombined, constitutively-expressed cilia reporters. The data reveal that only a subset of SOX9- positive astrocytes in the PFC possess cilia throughout development. Our work highlights the utility of Cre-Lox systems to target specific cell types and the importance of carefully validating genetic models.

Primary cilia function as specialized signaling centers that regulate many cellular processes including neuron and glia development. Astrocytes possess cilia, but the function of cilia in astrocyte development remains largely unexplored. Crucially, dysfunction of either astrocytes or cilia contributes to the molecular changes observed in neurodevelopmental disorders. Here, we show that a subpopulation of developing astrocytes in the prefrontal cortex are ciliated. This population corresponds to proliferating astrocytes and largely expresses the ciliary protein ARL13B. Genetic ablation of astrocyte cilia in vivo at two distinct stages of astrocyte development results in changes to Sonic Hedgehog (Shh) transcriptional targets. We show that Shh activity is decreased in immature and mature astrocytes upon loss of cilia. Furthermore, loss of cilia in immature astrocytes results in decreased astrocyte proliferation and loss of cilia in mature astrocytes causes enlarged astrocyte morphology. Together, these results indicate that astrocytes require cilia for Shh signaling throughout development and uncover functions for astrocyte cilia in regulating astrocyte proliferation and maturation. This expands our fundamental knowledge of astrocyte development and cilia function to advance our understanding of neurodevelopmental disorders.

ARL13B is a regulatory GTPase enriched in cilia, making it a popular marker for this organelle. Arl13b ^hnn/hnn mice lack ARL13B expression, die during midgestation, and exhibit defects in ciliogenesis. The R26Arl13b-Fucci2aR biosensor mouse line directs the expression of fluorescently tagged full-length Arl13b cDNA upon Cre recombination. To determine whether constitutive, ubiquitous expression of ARL13B-Cerulean can replace endogenous gene expression, we generated Arl13b ^hnn/hnn animals expressing ARL13B-Cerulean. We show that Arl13b ^hnn/hnn ;Arl13b-Cerulean mice survive to adulthood with no obvious physical or behavioral defects, indicating that the fluorescently tagged protein can functionally replace the endogenous protein during development. However, we observed that rescued males failed to sire offspring, revealing a role for ARL13B in spermatogenesis. This work shows that the R26Arl13b-Fucci2aR mouse contains an inducible allele of Arl13b capable of functioning in most tissues and biological processes.

Astrocyte cilia are largely understudied due to the lack of available tools. Astrocyte research advanced with the establishment of Aldh1l1-Cre ^ERT2 , an inducible Cre line that specifically targets the astrocyte lineage. Here, we develop and compare genetic models that label astrocyte cilia in the developing prefrontal cortex (PFC) using Aldh1l1-Cre ^ERT2 and Cre-dependent cilia reporters. We evaluate these models by testing different tamoxifen-induction protocols and quantifying the percentage of astrocytes labeled with the cilia reporters. We show that tamoxifen dosage impacts the expression of cilia reporters in astrocytes. We validate the maximum cilia-labeling efficiency of tamoxifen using constitutively-expressed cilia reporters. The data reveal that only a subset of SOX9- positive astrocytes in the PFC possess cilia throughout development. Our work highlights the utility of Cre-Lox systems to target specific cell types and the importance of carefully validating genetic models.