The Hedgehog (Hh) signaling pathway is essential for embryogenesis in a wide variety of organisms. Initially discovered in Drosophila where there is a single Hh ligand, the core components of the Hh pathway are conserved in vertebrates (Nüsslein-Volhard and Wieschaus, 1980). These include the vertebrate Hh receptor Patched1 (Ptch1), the obligate transducer of the pathway Smoothened (Smo), as well as the Gli transcription factors (Ci in Drosophila) that act as both activators (GliA) and repressors (GliR) to control target gene transcription (Briscoe and Thérond, 2013). There are three Hh ligands in vertebrates, Sonic (Shh), Indian (Ihh) and Desert (Dhh), that regulate a multitude of developmental processes including formation of the limbs and digits, the bones of the skull and face, and the patterning of the neural tube (Placzek and Briscoe, 2018). Diminished Hh signaling during embryogenesis results in birth defects whereas increased Hh signaling leads to tumors, highlighting the importance of the pathway and its regulation (Raleigh and Reiter, 2019).

Given the deep homology between invertebrate and vertebrate Hh signaling, the discovery that primary cilia are required for vertebrate, but not invertebrate, Hh signaling was unexpected (Huangfu et al., 2003; Huangfu and Anderson, 2006). Vertebrate Hh components dynamically traffic within primary cilia in response to Hh ligand (Corbit et al., 2005; Haycraft et al., 2005; Rohatgi et al., 2007). In the absence of ligand, Ptch1 is enriched on the ciliary membrane and Smo is barely detectable (Corbit et al., 2005; Rohatgi et al., 2007). Furthermore, full length Gli proteins traffic to the ciliary tip and back to the cytoplasm before being cleaved to their repressor form, which actively shuts down Hh target gene transcription in the nucleus (Kim et al., 2009; Liu et al., 2005; Wen et al., 2010; Humke et al., 2010). In contrast, upon ligand stimulation (Shh, Ihh or Dhh), the Ptch1 receptor binds the ligand and shuttles out of cilia (Rohatgi et al., 2007). Subsequently, Smo is enriched in the cilium and is subsequently activated (Kong et al., 2019; Corbit et al., 2005). Activated Smo promotes the processing of full-length Gli transcription factors into GliA, which turns on target genes (Aza-Blanc et al., 2000; Ruiz i Altaba, 1998). Ablation of cilia results in an absence of GliA and GliR production, rendering the pathway inert and leading to an absence of transcriptional response (Huangfu and Anderson, 2005). The dynamic ciliary movement of Shh components appears to be critical to pathway function, as alterations to cilia disrupt pathway output (Caspary et al., 2007; Huangfu and Anderson, 2006; Liem et al., 2012; Liem et al., 2009; Goetz et al., 2009; Murdoch and Copp, 2010; Tuz et al., 2014; Cortellino et al., 2009; Houde et al., 2006; Liu et al., 2005; Tran et al., 2008; Taylor et al., 2015).

Given that the fundamental logic of the pathway is conserved from flies through vertebrates, and that flies transduce Hh signals without relying on primary cilia, both evolutionary and mechanistic questions are raised as to how vertebrate cells co-opted the primary cilium for Hh signal transduction. One distinction lies in the fact that vertebrates use Hh signaling over a longer distance than flies, leading to the proposal that the primary cilium is a critical part of a mechanism underlying long range signaling (Bangs et al., 2015). For example, in neural patterning Shh is initially expressed in the notochord and is secreted to specify fates more than 20 cells away (Chiang et al., 1996; Briscoe et al., 2001; Roelink et al., 1994). At the evolutionary level, comparisons among organisms with cilia and or Hh have provided some clues. The round worm C. elegans have cilia yet do not possess Hh signaling as they don’t have most of the genes encoding the core components of Hh signal transduction (The C. elegans Sequencing Consortium, 1998; Roy, 2012). Curiously, a few components of Hh signaling such as fused and costal2 are in the C. elegans genome where they are functionally important for ciliogenesis (Ingham et al., 2011). Additionally, C. elegans retained a Ptch1 homolog important for development and pattern formation, but no Hh or Smo (Zugasti et al., 2005; Kuwabara et al., 2000). In contrast, planaria flatworms possess both cilia and Hh signaling but the cilia are not required to transduce Hh signaling (Rink et al., 2009). The first known evolutionary link between cilia and Hh is in sea urchins which transduce Hh signal in developing muscle tissue via motile cilia (Warner et al., 2014; Sigg et al., 2017). Subsequently, in vertebrates Hh signaling requires primary cilia. These data suggest that the mechanistic link of cilia and Hh is limited to deuterostomes and raises the question of whether the relationship of Hh and primary cilia originated near the last common ancestor of vertebrates, the urochordates.

ARL13B is a member of the ARF family of regulatory GTPases and is highly enriched on the ciliary membrane (Caspary et al., 2007). In mice, a null mutation of Arl13b leads to short cilia and to alterations in Shh signal transduction (Caspary et al., 2007; Larkins et al., 2011). ARL13 is ancient, predicted to be present in the last common eukaryotic ancestor. ARL13 appears to have been lost during evolution in organisms that lack cilia and duplicated to ARL13A and ARL13B in the urochordates, thus ARL13B is proposed to hold important clues in deciphering the links between primary cilia and vertebrate Hh signaling (Schlacht et al., 2013; Li et al., 2004; Kahn et al., 2008; East et al., 2012; Logsdon, 2004).

ARF regulatory GTPases, like ARL13B, are best known to play roles in membrane trafficking (D'Souza-Schorey and Chavrier, 2006). As is true for a large number of regulatory GTPases, ARL13B is functionally diverse (Sztul et al., 2019). It regulates endocytic traffic (Barral et al., 2012), as well as the phospholipid composition of the ciliary membrane through recruitment of the lipid phosphatase INPP5E to the ciliary membrane (Humbert et al., 2012). ARL13B also has a conserved role as a guanine nucleotide exchange factor (GEF) for ARL3, another ciliary ARF-like (ARL) protein (Gotthardt et al., 2015; Zhang et al., 2016; Hanke-Gogokhia et al., 2016; Ivanova et al., 2017). ARL13B regulates intraflagellar transport (IFT), the process that builds and maintains cilia (Cevik et al., 2010; Li et al., 2010; Nozaki et al., 2017). It is known to interact with several proteins associated with cilia, including the exocyst, tubulin and UNC119 (Seixas et al., 2016; Zhang et al., 2016; Larkins et al., 2011; Revenkova et al., 2018). Critical to this work, loss of ARL13B disrupts Shh signal transduction in at least two distinct ways: Smo enrichment in cilia occurs even in the absence of ligand and Gli activator production is diminished, although Gli repressor is made normally (Caspary et al., 2007; Larkins et al., 2011).

Due to the high enrichment of ARL13B on the ciliary membrane, ARL13B is assumed to function in its diverse roles from within the cilium. However, ARL13B is present in early endosomes and circular dorsal ruffles on the cell surface (Barral et al., 2012; Casalou et al., 2014). We previously showed that a V358A variant of ARL13B does not localize to cilia as it disrupts a known VxPx cilia localization sequence (Higginbotham et al., 2012; Mariani et al., 2016). Exogenous overexpression of a ARL13B^V358A construct in Arl13b null cells does not rescue ARL13B-dependent phenotypes such as cilia length as well as interneuron migration and connectivity, consistent with ciliary ARL13B mediating these processes (Higginbotham et al., 2012; Mariani et al., 2016). In contrast, we found that overexpressed ARL13B^V358A does rescue the Shh-dependent ciliary enrichment of Smo in mouse embryonic fibroblasts, arguing that ARL13B may function outside the cilium to regulate Smo traffic (Mariani et al., 2016). Together, these results raise the question of where ARL13B functions.

To define where ARL13B functions in relation to cilia, we wanted an in vivo model so generated mice carrying the ARL13B^V358A point mutation using CRISPR/Cas9. Here we demonstrate that ARL13B^V358A protein was undetectable in Arl13b^V358A/V358A cilia in both neural tube and mouse embryonic fibroblast cilia, even after blocking retrograde ciliary traffic. We report that Arl13b^V358A/V358A mice were viable, fertile, and transduced Shh signal normally. We found ARL3 and INPP5E did not localize to the short cilia present in/on Arl13b^V358A/V358A cells. These data indicate that ARL13B’s roles within and outside cilia can be uncoupled; ARL13B’s role in regulating cilia length is from within cilia, whereas its control of Shh signaling appears to be from outside the cilium. Thus, these data imply that the cilia defects seen in the complete absence of ARL13B do not underlie the alterations in Shh transduction, which is unexpected given the requirement of cilia for Shh signal transduction.

Here we show that ARL13B’s role(s) in cell ciliation and cilia length, along with the ciliary enrichment of a subset of proteins, can be uncoupled from ARL13B’s function in regulating Shh signal transduction (Figure 7). Furthermore, we demonstrate this functional distinction correlates with ARL13B spatial localization to the cilium. By generating a mouse expressing a cilia-excluded variant of ARL13B from the endogenous locus, we showed ARL13B^V358A protein is not detectable in cilia of embryonic neural tube or MEFs, even upon retrograde ciliary dynein traffic blockade. Furthermore, we detected 30% less ARL13B protein overall in Arl13b^V358A/V358A embryos compared to control embryos. While this reduction is statistically significant, it is unclear whether it is biologically significant given that Arl13b^hnn/hnn null mutations are recessive and no Arl13b^hnn/+ heterozygous phenotype is reported (Caspary et al., 2007; Larkins et al., 2011).

Figure 7

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In contrast to the E13.5 lethality of Arl13b^hnn/hnn embryos, we found Arl13b^V358A/V358A mice were viable and fertile with correct patterning of the neural tube, indicating normal Shh signal transduction. These results are consistent with our data showing that ARL13B^V358A protein retains all known biochemical function including GEF activity (Mariani et al., 2016; Ivanova et al., 2017). However, we observed several Arl13b^hnn/hnn phenotypes in Arl13b^V358A/V358A cells, including loss of ARL3 and INPP5E ciliary enrichment, along with low percentage of ciliated cells and shorter average cilia length. Taken together, our data show that despite the normal high ciliary enrichment of wild type ARL13B and the requirement of cilia for Shh signaling, cilia-excluded ARL13B^V358A is sufficient for Shh signal transduction.

We regard ARL13B^V358A as cilia-excluded based on several lines of evidence. We did not detect ciliary ARL13B^V358A in vivo or in vitro using any of five validated ARL13B antibodies. These antibodies are against two antigens, the full-length protein or residues 208–427, and were independently generated so are likely to recognize a number of epitopes. Given that four of five antibodies are polyclonal and ARL13B^V358A displays normal GTP binding, intrinsic and GAP-stimulated GTP hydrolysis, and ARL3 GEF activity, it is likely that the ARL13B^V358A protein retains the wild type structure enabling antibody recognition. Indeed, we observed little or no loss in sensitivity in detecting protein in the immunoblot assays. Furthermore, we could not forcibly enrich ciliary ARL13B^V358A through retrograde transport blockade with ciliobrevin-D nor could we detect any ciliary enrichment of ARL13B^V358A upon overexposure of the relevant fluorescent channel. Alternative ways of trafficking proteins out of cilia include BBSome-dependent or diffusion-dependent mechanisms raising the possibility that ARL13B is trafficked via dynein-independent mechanisms. The fact that we observed ciliary phenotypes, namely short cilia and abnormal ciliary ARL3 and INPP5E localization in ARL13B^V358A expressing cells, argue that ARL13B normally performs these functions from within cilia (Caspary et al., 2007; Larkins et al., 2011; Zhang et al., 2016; Humbert et al., 2012; Nozaki et al., 2017). While we cannot exclude the possibility that sub-detectable levels of ARL13B^V358A are present and functional in cilia, we note such a level would need to be sufficient for Shh signaling yet insufficient for ARL3 or INPP5E ciliary localization along with proper regulation of cilia length.

Our data support ARL13B regulating different biological processes from its distinct subcellular localizations consistent with how other GTPases act from multiple sites in cells through different effectors. ARL13B^V358A disrupts cilia localization of INPP5E and ARL3, but not Shh components and thus provides a novel tool with which to identify additional ARL13B effectors. Such effectors may also inform us as to what subcellular compartment ARL13B functions from as the cellular ARL13B antibody staining we observe is inconclusive. It is surprising that ARL13B^V358A is sufficient to regulate Shh signaling because cilia are required for Shh signal transduction and because ARL13B is highly enriched in cilia (Caspary et al., 2007; Huangfu et al., 2003). These observations suggested that the ciliary defects observed in Arl13b^hnn/hnn mutants caused the Shh defects. However, our data indicate that ARL13B regulates cilia length and Shh signaling through distinct localization. Thus, the cilia defects in Arl13b^hnn/hnn mutants do not underlie Shh misregulation.

Based on the normal cilia trafficking of Shh components in Arl13b^V358A/V358A mutants and the abnormal cilia trafficking of Shh components in the complete absence of ARL13B (Larkins et al., 2011), ARL13B likely regulates Shh signaling from the cell body by controlling Shh component traffic to and/or from the primary cilium. Both Smo and ARL13B traffic is linked to endosomes providing one possible non-ciliary organelle (Barral et al., 2012; Wang et al., 2009; Milenkovic et al., 2009). Arl13b^hnn/hnn cells display constitutive Smo ciliary enrichment along with little enrichment of Gli proteins at the ciliary tip (Larkins et al., 2011). Our data do not distinguish between ARL13B playing direct roles in traffic of multiple Shh components or whether the normal Smo ciliary enrichment with ARL13B^V358A subsequently causes normal cilia traffic of downstream components. ARL13B regulates a step downstream of activated Smo that controls transcription factor Gli activation, but not repression (Caspary et al., 2007; Bay et al., 2018). We argue from our results that this step is also intact in the presence of ARL13B^V358A.

The fact that ARL13B^V358A can mediate normal ciliary Smo enrichment is especially interesting given that ARL3 and INPP5E localization to cilia is compromised by this mutation. This suggests that ARL13B controls cilia enrichment via multiple localizations and effectors. This is consistent with our understanding of ARF family members, as they are known to perform multiple tasks from different sites within a cell (Sztul et al., 2019). We speculate that ARL3 residence in cilia may require that ARL3 be in (or at least cycle through) its activated, GTP-bound conformation as ARL13B^V358A retains its ARL3 GEF activity, but not its cilia localization. ARL13B is in a common protein complex with INPP5E so absence of ARL13B from cilia may disrupt formation or maintenance of the complex there (Humbert et al., 2012; Nozaki et al., 2017). INPP5E regulates Shh signaling through regulation of the phosphoinositol composition of the ciliary membrane. INPP5E loss results in increased ciliary PIP₂ and enrichment of Shh repressors in cilia thus resulting in lowered Shh response (Garcia-Gonzalo et al., 2015; Chávez et al., 2015; Dyson et al., 2017). It is not clear why the absence of ciliary INPP5E in ARL13B^V358A cells does not lead to aberrant Shh signal transduction.

In Arl13b^hnn/hnn embryos and MEFs, cilia are shorter than normal and have a microtubule defect where the B-tubule of the microtubule outer doublet is open (Caspary et al., 2007; Larkins et al., 2011). Consistent with this, we observed shorter cilia in Arl13b^V358A/V358A MEFs. Similarly, we observed a comparable reduction in the percent of cilia in Arl13b^hnn/hnn and Arl13b^V358A/V358A MEFs compared to wild type MEFs. While loss of ARL13B results in short cilia, increased expression of ARL13B promotes cilia elongation so it is not yet clear what ARL13B’s role is in controlling cilia length (Lu et al., 2015; Hori et al., 2008; Caspary et al., 2007; Larkins et al., 2011). Additionally, it is unclear whether the mechanism from within cilia through which ARL13B controls length or percent of ciliated cells is the same, or distinct from, the mechanism through which ARL13B regulates ARL3 or INPP5E residence in cilia.

Perhaps the most intriguing implication of our data pertains to understanding the evolution of cilia and Hh signaling. ARL13 functions in cilia formation and maintenance in Chlamydomonas and C. elegans, neither of which have Hh signaling, consistent with the ancient role of ARL13 controlling ciliation and cilia length (Cevik et al., 2010; Cevik et al., 2013; Stolc et al., 2005; Miertzschke et al., 2014). Our data support ARL13B retaining ARL13’s ancient role in ciliogenesis. However, our data show that ARL13B does not work from within the cilium to regulate Shh component ciliary traffic or Shh signal transduction. As an ARL protein involved in membrane traffic, we speculate that ARL13B may have linked Shh component trafficking to the primary cilium, albeit from outside the cilium. That the ARL13 duplication could relate to the mechanism linking primary cilia and Hh signaling within the deuterostome lineage is a possibility worth exploring.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Eduardo D Gigante

   1. Neuroscience Graduate Program, Emory University School of Medicine, Atlanta, United States
   2. Department of Human Genetics, Emory University School of Medicine, Atlanta, United States

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1486-5377

2. Megan R Taylor

   Emory College of Arts and Sciences, Emory University School of Medicine, Atlanta, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

3. Anna A Ivanova

   Department of Biochemistry, Emory University School of Medicine, Atlanta, United States

   Contribution

   Formal analysis, Validation, Investigation, Visualization

   Competing interests

   No competing interests declared

4. Richard A Kahn

   Department of Biochemistry, Emory University School of Medicine, Atlanta, United States

   Contribution

   Supervision, Funding acquisition, Visualization, Methodology

   Competing interests

   No competing interests declared

5. Tamara Caspary

   Department of Human Genetics, Emory University School of Medicine, Atlanta, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Project administration

   For correspondence

   tcaspar@emory.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6579-7589

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