Primary cilia deficiency in neural crest cells causes Anterior Segment Dysgenesis

During eye embryogenesis, neural crest cells (NCC) of the periocular mesenchyme (POM) migrate to the anterior segment (AS) of the eye and then differentiate into the corneal stroma and endothelium, ciliary body, iris stroma, and the trabecular meshwork. Defective development of these structures leads to anterior segment dysgenesis (ASD) that in 50% of the cases leads to glaucoma, a leading cause of blindness. Here, we show that the primary cilium is indispensable for normal AS development and that its ablation in NCC induces ASD phenotypes including; small and thin cornea, impaired stromal keratocyte organization, abnormal iridocorneal angle with reduced anterior chamber and corneal neovascularization. These defects are similar to those described in patients with AS conditions such as Axenfeld-Rieger syndrome and Peter’s anomaly. Mechanistically, disruption of the primary cilium in the NCC resulted in reduced hedgehog (Hh) signaling in the POM, canonically activated by the Indian Hedgehog ligand expressed by endothelial cells of the choroid. This caused decreased cell proliferation in a subpopulation of POM cells surrounding the retinal pigmented epithelium. Moreover, primary cilium ablation in NCC also led to a decreased expression of Foxc1 and Pitx2, two transcription factors identified as major ASD causative genes. These findings suggest that primary cilia are indispensable for NCC to form normal AS structures via Hh signaling. Defects in primary cilia could, therefore, contribute to the pathogenesis of ASD, and to their complications such as congenital glaucoma.


INTRODUCTION
Anterior segment dysgenesis (ASD) is a term referring to a spectrum of congenital disorders of structures of the anterior segment (AS) of the eye. Abnormalities typically associated with ASD include; corneal opacity, cataract, posterior embryotoxon, iris hypoplasia, corectopia or polycoria, adhesions between iris and cornea or lens and cornea [1]. Approximately 50% of ASD patients develop glaucoma that can lead to visual impairment and blindness [1][2][3][4]. Although mutations in genes encoding transcription factors, transporters, and glycosylating proteins have been described in patients with ASD, several ASD cases still await genetic elucidation [1][2][3][4].
Moreover, the cellular and molecular mechanisms underlying the pathogenesis of different ASD conditions remain unknown. Most AS structures are derived from the neural crest cells (NCC), including the corneal stroma and endothelium, ciliary body muscle and body, iris stroma, and the trabecular meshwork [5]. In mice, migrating NCC of the periocular mesenchyme (POM) begin to invade the AS of the eye between E11.5 and E12.5, and differentiate into corneal endothelial cells and keratocytes [6,7]. By E16.5, the presumptive iris is visible and detaches from the cornea, while the drainage structures (trabecular meshwork and Schlemm's canal) continue to develop postnatally [8]. NCC also give rise to the sclera, pericytes of the choroid and hyaloid vasculature, orbital cartilage and bone, and oculomotor tendons [9]. The neural crest is a transient embryonic structure in vertebrates that delaminates from the border between the neural plate and the non-neural ectoderm; NCC migrate throughout the embryo to multiple locations and differentiate into a wide variety of cell types and tissues [10,11]. Recent studies have shown that primary cilia play essential roles in morphogenetic processes involving neural crest-derived cells such as craniofacial development [12,13]. It has been proposed that primary cilia seem to mediate tissue-tissue interactions requiring reciprocal signaling rather than purely NCC specification [14,15].
Primary cilia are microtubule-based cellular organelles that emanate from the basal body and extend from the plasma membrane. A bidirectional movement of protein particles along the axoneme, called intraflagellar transport (IFT), ensures the appropriate assembly and maintenance of cilia [16][17][18][19][20]. Primary cilia play a pivotal role in the development and tissue homeostasis by regulating the Hedgehog (Hh), Wnt, and Notch signaling pathways among others [21,22]. Hh is the pathway the most strongly associated with the primary cilium [23,24].
The binding of one of the three mammalian Hh ligands (Sonic hedgehog (SHH), Indian hedgehog (IHH) or Desert hedgehog (DHH)) on the receptor Patched (PTCH) induces the exit from the cilium of PTCH and an accumulation of the Hh transducer Smoothened (SMO) in the cilium. Subsequently, SMO activates the Gli transcription factors which translocate in the nucleus and activate the expression of Hh target genes [25]. Target genes of the Hh pathway are involved in cell proliferation, maintenance of stemness, cell-fate determination, cell survival and epithelial to mesenchymal transition [26].
Dysfunctions of cilia lead to a wide range of human diseases called ciliopathies, that affect most human organ systems [24]. Conditions of the AS, including ASD, were described associated with ciliopathies. Patients affected by Meckel syndrome, a severe ciliopathy, present AS abnormalities including microphthalmos/anophthalmos, aniridia, cryptophthalmos, sclerocornea, abnormal corneal thickness, corneal neovascularization, abnormal iridocorneal angle, tunica vasculosa lentis, and persistence of nuclei in lens fibers [27]. Cases of microphthalmia were recently reported in patients affected by oral-facial-digital syndrome [28,29]. Corneal opacity has been detected in a patient affected by Joubert syndrome [30], glaucoma and cataract are common conditions associated with Bardet-Biedl and Lowe syndromes [31,32], and cataract and keratoconus with Leber's congenital amaurosis [31]. Moreover, one of the features of the Biedmond syndrome type 2, resembling to the Bardet-Biedl syndrome, is the presence of iris colloboma (OMIM #210350).
Interestingly, systemic mutations in ciliogenic genes causing an increase of the Hh activity and a deletion of Gli3, which acts predominantly as a repressor of the Hh target genes, lead to similar abnormal ocular development [38][39][40][41][42]. On the other hand, the loss or downregulation of the Hh activity leads to severe craniofacial and ocular defects including anophthalmia, cyclopia, microphthalmia, and coloboma [9]. This leads us to hypothesize that the primary cilium plays a pivotal role in the development of the AS and that dysfunction of the primary cilium in NCC could lead to conditions similar to those associated with ASD.
In this study, we investigated the role of the primary cilium in the development of the NCC derived ocular structures and its possible role in ASD. We showed that NCC of the POM are ciliated and that primary cilia persist in keratocytes of adult mice. We demonstrated that ablation of the primary cilium in NCC induces an ASD phenotype with impaired corneal stroma organization and dimension, abnormal iridocorneal angle and corneal neovascularization.
We also showed a reduction of the Hh signaling pathway specifically in a subset of cells in the POM surrounding the retinal pigmented epithelial cells (RPE). Furthermore, we identified the endothelial cells of the choroid as the cells expressing Ihh, which is the Hh ligand maintaining the signaling activity in the POM surrounding the RPE layer in normal eye development.

Primary cilium ablation in NCC leads to ASD phenotype
To determine spatiotemporal distribution of primary cilia in NCC-derived tissue of the POM we visualized cilia by immunofluorescence (IF) using an anti-Arl13b Ab, a widely accepted ciliary marker [43] in genetically labeled NCC. NCC were traced by using the Rosa26 mT/mG (mT/mG) reporter mouse line [44] crossed to the Wnt1-Cre mouse [45] in which the Cre recombinase is under the control of the Wnt1 promoter, a gene highly expressed in early stages of NCC specification. In the Wnt1-Cre;mT/mG transgenic line, the Cre-dependent excision of a cassette expressing the red-fluorescent membrane-targeted tdTomato (mT) allows the expression of a membrane-targeted green fluorescent protein (mG) in bona fide NCC-derived tissues ( Figure   1A). We observed that at E14.5, all NCC-derived tissues of the POM and the presumptive corneal stroma were ciliated ( Figure 1A). Our previous studies reported that while primary cilia are present in developing corneal endothelium (also a NCC-derived tissue), they disassemble in adult corneal endothelium at steady state [46]. To assess the presence/absence of primary cilia in adult corneas we utilized a transgenic mouse line expressing the ciliary membrane protein somatostatin receptor 3 fused to GFP under the ubiquitous promoter for actin (Sstr3::GFP) [47].
Intravital microscopy revealed that cilia were present in all keratocytes of the corneal stroma of 3-month-old mice ( Figure 1B). To gain ultrastructural insights we analyzed corneal stroma and POM in developing and adult eyes. TEM showed that in developing eyes, cilia emanated from the cellular surface into the extracellular matrix whereas, cilia of newborn keratocytes appeared to be intracellular or largely invaginated in a long ciliary pocket with their axis parallel to the cell plane ( Figure 1D-E). Interestingly, the tip of cilia in developing corneas and POM were observed to interact with cellular protrusions of the neighboring cells (Figure 1C-D). Moreover, the plasma membrane of the cellular protrusion at the contact point with the ciliary tip appeared to be highly electron-dense, suggesting the presence of protein components or modified lipids in this region (Figure 1D-E). To investigate the involvement of primary cilia in AS development we set out to ablate Ift88, a subunit of the IFT machinery required for cilia assembly and maintenance [48], in NCC. To do so, we generated the Wnt1-Cre;Ift88 fx/fx mouse (cKO) which was indistinguishable from the null hemizygous Wnt1-Cre;Ift88 fx/-. In this mouse the Ift88 gene is excised in all migrating mesenchymal cells expressing Wnt1 leading to complete ablation of the primary cilium (Supplement Figure 1A) [45,49]. To monitor ablation of cilia in the NCC of the POM we generated the Wnt1-Cre;IFT88 fx/fx ;mT/mG mouse and we labeled cilia with an anti-Arl13b Ab [43]. In control mice, virtually all the NCC of the POM appeared ciliated during development (Fig. 1A). In contrast, in the Wnt1-Cre;IFT88 fx/fx ;mT/mG mutant mice cilia were absent in most of the POM cells expressing Cre (Supplement Figure 1B). We confirmed ablation of cilia in keratocyte precursors by TEM. In keratocyte precursors, the basal body apparatus was observed to reach the apical plasma membrane in both control and mutant corneas, however, primary cilia were only observed emanating from basal bodies of control keratocytes (Supplement Figure 1C). Wnt1-Cre;Ift88 fx/fx mutant mice died at birth and E18.5 embryos displayed strong craniofacial defects including increased frontal width, wider frontonasal prominence and increase of the distance between the nasal pits, consistent with previous studies (Figure 2A) [36,37]. In addition, we detected abnormalities in the anatomy of the eye. At E14.5, a developmental stage preceding the closure of the eyelids, the axis of the eyeballs was misaligned facing downward in the cKO whereas, in control it remained perpendicular to the sagittal plane of the head (Figure 2A). Furthermore, the outline of the presumptive iris appeared irregular in the mutant while in the control it described a nearly perfect circle (Figure 2A). Later in development (E18.5), the irregularities of the iris exacerbated and the developing cornea appeared smaller than that of the control, strongly arguing toward severe defects of the AS including a reduced anterior chamber (Figure 2). Thus, these macroscopic anatomical abnormalities suggest a morphogenetic condition of the AS in the eye of the mutant.
To further characterize the mutant phenotype of the AS, we conducted histological analysis of paraffin and plastic embedded samples (Figure 2). The eye field of the cKO embryos at E10.5 and 14.5 appeared indistinguishable from that of the control ( Figure 2B). In contrast, at E15.5, we observed a significant reduction of the anterior chamber in the cKO (Figure 2B-D). At this stage, the mutant was also lacking most of the mesenchymal cells condensing at the developing iridocorneal angle between the cornea and the presumptive iris that was instead clearly visible in the control ( Figure 2D). As a result, the iridocorneal area appeared disorganized in the mutant with a narrower angle than in the control. Between E16.5 and E18.5, the corneal stroma thickness and the corneal diameter were both significantly reduced in the cKO embryo compared to the control (Figure 2B-C). Moreover, the iridocorneal angle abnormalities found in the mutant eye persisted with the presumptive iris significantly shorter than that of the control ( Figure 2D). Plastic cross sections of the cornea also revealed that the density of the keratocytes was significantly higher in the cKO stroma compared to control however, the total number of cells in corneal stroma remained unchanged in both genotypes ( Figure 2E). Thus, the increased keratocyte density detected in the cKO embryos was essentially due to its smaller volume cornea compared to that of control. Moreover, the unchanged number of keratocytes in the stroma of both genotypes suggests that the primary cilium ablation did not impair the NCC migration into the corneal stroma. Thus, the ablation of the primary cilium in the NCC leads to ASD that is not due to NCC migration defects.
Next, we have examined the tridimensional organization and the morphology of keratocytes.
Keratocyte morphology and spatial distribution across the stroma vary in mammals, with an increased density, flattening, and extension of the keratocytes in the posterior part of the stroma in late embryonic development [50,51]. To determine stromal organization, we developed a quantitative tool using live confocal imaging on mT/mG mice. Segmentation and quantification of the stromal extracellular spaces was carried out with ilastik [52] (Supplement Figure 2). We determined the amount (%) and the average size of segmented extracellular spaces using Fiji [53]. In control embryos, the amount of extracellular spaces was significantly lower in the posterior than the anterior portion of the corneal stroma, defining an antero-posterior gradient of the keratocyte density ( Figure 3B). Moreover, the size of the extracellular spaces in the posterior part was significantly smaller than in the anterior part of the stroma in control embryos, defining an antero-posterior gradient of the extracellular space size ( Figure 3C). In cKO embryos, the antero-posterior gradient of the keratocyte density was maintained. However, that of the average size of extracellular spaces was not ( Figure 3B). In addition, the amount of extracellular spaces was significantly reduced in both the anterior and posterior parts of the cKO stroma compared to control (Figure 3B-C). Thus, the keratocyte distribution in the corneal stroma of the cKO embryos was denser than that of the control. Moreover, the differences in the anterior-posterior gradient of the amount of extracellular space and the average size of single extracellular spaces are abnormal in the cKO suggesting a defective tridimensional organization of the keratocytes in the mutant. Thus, the ablation of the primary cilium in NCC impairs the spatial organization of keratocytes in the stroma independently from cell migration.
Because the density and the spatial organization of the keratocytes are abnormal in the mutant stroma we sought to investigate the morphology of single keratocytes in vivo. Keratocytes are characterized by their cytoplasmic processes interconnected with each other to form a dense and complex 3D network [54]. We focused on the junction between the corneal epithelium and the stroma because here the density of keratocytes is lowest. In both genotypes, we could distinguish single cytoplasmic processes of the first keratocyte layer expressing the mG reporter underneath the overlying corneal epithelial cells layer expressing the mT reporter. The number of cytoplasmic processes was significantly increased in the cKO embryos compared to control ( Figure 3D-E), suggesting a possible role of the primary cilium in controlling the morphology and the number of cytoplasmic processes of keratocytes.

Ablation of Ift88 in NCC disrupts Hh signaling in a subpopulation of POM cells surrounding RPE and at the iridocorneal angle
Morphological changes, as well as spatial organization of the cells in a tissue, occur as cells differentiate. This implies a wide variety of signaling pathways occurring in a timed and coordinated fashion. Mice lacking heparan sulfate in NCC display ASD phenotypes resembling those caused by the lack of NCC cilia including cornea stroma hypoplasia, dysgenesis of the iridocorneal angle and decreased depth of the anterior chamber [55]. Because in these mice the TGFβ2 pathway is disrupted, we assessed whether the primary cilium ablation in the NCC affected the TGFβ2 pathway in the cornea. At E18.5, the expression of Tgfbr1, Tgfbr2, Smad2, Smad3, Smad4 and Smad7 and the percentage of pSmad2/3 + cells in the cornea remained indistinguishable between the cKO and control mice (Supplement Figure 3).
Hh, Wnt and Notch signaling pathways have been linked to the primary cilium [22]. We therefore used quantitative real-time PCR (RT-qPCR) on isolated ocular NCC and reporter mouse lines to examine the expression of specific target genes of the above-mentioned pathways. To isolate ocular NCC, cKO and control mice were crossed to the mT/mG transgenic reporter line (Supplement Figure 1) and mG + cells of the dissected sclera and cornea tissue were digested and FACS sorted following the approach shown in (Figure 4A). NCC from the E18.5 cKO mouse showed a significant decrease of Hh target genes including Gli1 and Ptch1 but not  Figure 5A). In addition, we also arbitrarily defined a Hh-negative area of the POM encompassed between 20 µm and 50 μm from the RPE cell layer (20-50 µm) functioning as an internal control together with the Hh-negative cornea. Proliferation rates were assessed by counting the number of cells stained with an anti-Ki67 Ab and normalized to the total number of cells in the same area stained with DAPI. At E14.5, cell proliferation was significantly decreased in the POM close to the RPE layer (0-20µm) of the cKO mutants compared to control ( Figure 5B). In contrast, proliferation rates remained unchanged in the outer region of the POM (20-50 µm) and in the cornea where the Hh was normally not activated ( Figure 5B). Thus, ablation of the primary cilium in NCC leads to a decreased cell proliferation specifically in a subpopulation of POM cell surrounding the RPE due to local inactivation of the Hh signaling.
Embryonic POM cells of the sclera progressively lose their ability to respond to Ihh produced by choroidal endothelial cells Next, we sought to determine how the Hh signaling is maintained in a restricted subpopulation of NCC within the POM. Because mammals express three different Hh homologues, Dhh, Ihh and Shh, we analyzed dissected tissue of the eye from E18.5 mice, including NCC by RT-qPCR to determine which Hh ligand is expressed in NCC derived tissue of the eye as shown in the scheme in Figure 6A. We found that the expression of Ihh was significantly higher than that of Dhh and Shh in the sclera including the RPE layer but not in the cornea ( Figure 6A). As control, we found that Shh but not Dhh and Ihh was expressed in the isolated neural retina where NCC are excluded ( Figure 6A).
To determine the source of the Hh ligand we sorted and analyzed by RT-qPCR different cell types of the sclera. A previous study showed by in situ hybridization and immunohistochemistry an overlapping staining between Ihh and collagen type IV, a vascular endothelial marker,  Figure 6B). At E18.5, among the non-NCC, Ihh was significantly more expressed by the choroidal endothelial cells than Dhh and Shh ( Figure 6C). Thus, we provided molecular evidence that endothelial cells of the POM produce the Hh ligand Ihh in the sclera.
As shown in Figure 4 during eye development, NCC of the peripheral POM progressively lost the Hh signaling activity while Hh-responsive cells remained confined to the choroid area and at the iridocorneal angle. To gain mechanistic understanding on how this process occurs we analyzed the presence of cilia and Hh components in the ciliary compartment of Hh-negative POM cells. In presence of an Hh ligand, its receptor PTCH exits the ciliary compartment allowing SMO to concentrate into the cilium, which is an essential step to activate the Hh signaling [25]. To determine whether POM cells were still ciliated and able to respond to a Hh signal, we stained primary cilia and SMO in whole mount preparations of the wild-type sclera.
We collected confocal optical sections of the POM between the RPE layer and the periphery of the sclera as indicated in (Figure 6D). At E14.5 and P0, all cells of the POM were ciliated ( Figure 6E). Thus, we excluded the possibility that NCC of the peripheral POM lost the Hh activity due to resorption of the primary cilium. At E14.5, we observed an accumulation of SMO in primary cilia of the inner cell layers of the POM surrounding the RPE (0-18 μm) consistent with the Hh activity detected in these cell layers (Figure 4). In contrast, we did not detect SMO in the primary cilium in cells of the peripheral POM (>18 μm from the RPE) (Figure 6E-F). At P0, SMO was undetectable in primary cilia of NCC of the entire POM (Figure 6E-F). However, when eyeballs from P0 mice were treated with SAG, SMO accumulated in cilia of POM cells ( Figure 6E-F). This implies that POM cells of the P0 sclera were still able to activate Hh signaling upon ligand stimulation, and suggests that the decreased Hh activity in the POM during ocular development is controlled by the diffusion in the POM of the Hh ligand Ihh.

Primary cilium ablation in NCC leads to corneal neovascularization
Heterozygote mutations in FOXC1 and PITX2 genes account for ~40% of ASD cases [60]. In mice, Foxc1 and Pitx2 haploinsufficiency leads to ocular phenotypes recapitulating human conditions of ASD including iridocorneal angle abnormalities, thinning and abnormal vascularization of the cornea [61][62][63][64]. Because these phenotypes were also detected in the cKO mouse, we tested whether absence of cilia in the NCC affected the expression of ASD genes in E18.5 cKO embryonic corneas. RT-qPCR revealed that gene expression of Foxc1 and Pitx2 was significantly decreased in corneas of cKO embryos compared to controls while Pax6 expression remained unchanged ( Figure 7A). Because FOXC1 and PITX2 are indispensable to specify corneal angiogenic privilege [1,[62][63][64] we examined the neovascularization process in the cKO mutant. To visualize blood vessels, we stained whole corneas with an Ab directed to endomucin, a marker of the vascular endothelium. Abnormal spread of blood vessels was detected into the corneas of E18.5 cKO mutant embryos (Figure 7B-C) where vessels covered about 8% of the total corneal area ( Figure 7D). To gain additional insights on the abnormal neovascularization process, we performed live imaging using confocal microscopy at the cornea periphery using enucleated eyes from E18.5 embryos with the mT/mG reporter. Due to their mesenchymal origin, blood vessels were easily detectable as red (mT) tubular network clearly distinct from the green (mG) NCC-derived POM (Supplement Figure 5). By confocal optical sectioning we identified the major arterial circle which served as reproducible reference between the end of the sclera and the beginning of the cornea. The average area occupied by blood vessels in microscope fields selected at the corneal periphery, occupied ~30% of the total area in the cKO but only ~4% in the control (Figure 7E-F). In addition, while peripheral blood vessels of the control were present only in the superficial layers of the sclera-cornea interface on both sides of the major arterial circle (~20% of the thickness), those of the mutant invaded the lower layers of both, the sclera (~50% of the thickness) and the cornea (~80% of the thickness).
These results demonstrate that ablation of the primary cilium in the NCC leads to the loss of the angiogenic privilege in the cornea as well as vascular abnormalities in the sclera, potentially due to the lowered expression of Foxc1 and Pitx2.

Primary cilium ablation in NCC impairs early corneal innervation and centripetal migration of the sensory nerves
Concurrent with the establishment of the angiogenic privilege is corneal innervation [65].
Because corneal sensory nerves are derived from the neural crest that is part of the trigeminal ganglion [66], we assessed whether primary cilium ablation in NCC would also impact the corneal innervation. During development, corneas of both control and cKO embryos were innervated ( Figure 8A). However, at E13.5, the number of nerve bundles detected in the cKO eye was significantly lower compared to that of the control (Figure 8B). Moreover, abnormally long nerve projections across the cornea were observed in cKO embryos whereas nerves are only visible at the periphery of the cornea in control (Figure 8A). At E18.5, corneas of both genotypes were fully innervated (Figure 8A), including the corneal epithelium (Supplement Figure 6). However, the corneal nerve density was significantly higher in the cKO embryos ( Figure 8C). In addition, the nerves in cKO corneas were less organized centripetally compared to the control. The average angle θ formed by the major nerve branches with the radius of the cornea was significantly bigger in cKO embryos compared to controls (Figure 8D-E). These data suggested that the primary cilium ablation in the NCC impaired the early corneal innervation and the centripetal migration of the sensory nerves.

DISCUSSION
Ocular conditions such as microphthalmos/anophthalmos, aniridia, sclerocornea, abnormal corneal thickness, corneal neovascularization, abnormal iridocorneal angle, corneal opacity, glaucoma, and cataract, have been reported in patients affected by ciliopaties including Joubert and Mekel syndrome, suggesting an implication of the primary cilium in AS development [27][28][29][30][31][32]. However, the pathogenesis of these conditions remains largely unknown. In the present study, we have demonstrated that conditional ablation of the primary cilium in NCC led to ASD with conditions similar to those observed in humans. We showed that primary cilia are present in virtually all neural crest-derived cells of the POM including keratocytes. However, we found including a large portion of AS structures [5]. Relevant to our study, conditional deletions of ASD causative genes such as Foxc1, Pitx2 or Ext1 in NCC, lead to defects of the AS including anophthalmos, abnormal corneal thickness, reduced anterior chamber, defective iridocorneal angle, and corneal neovascularization similar to abnormalities observed in ASD patients carring dominant mutations in these genes [55, 64,73]. However, although the cKO mice revealed a direct connection between the primary cilium and ASD, its severe craniofacial defects caused late embryonic death and prevented us to follow the postnatal development of structures such as the iris, trabecular meshwork and Schlemm's canal [8]. Thus, further studies involving conditional ablation of the cilium in specific AS tissues will be needful. The abnormal corneal neovascularization observed in the cKO could also derive from the reduction or absence of the Hh activity in the POM. In a zebrafish model, the loss of Hh signaling induces excess sprouting of the blood vessels in the dorsal eye and impaires growth of the blood vessels in the ventral eye [76]. These observations in the eye tissue of the fish are also consistent with the aberrant expansion of the blood vessels domain deep into the sclera of cKO embryos (Figure 7). A thorough analysis of the choroid in NCC conditional mutants with a reduced Hh activity obtained independently from the ablation of the primary cilium could elucidate a specific role of the Hh pathway in development and patterning of the choroid. The process of corneal innervation occurs in parallel with the establishment of the corneal angiogenic privilege. The majority of corneal nerves derive from the ophthalmic division of the trigeminal ganglion of NCC origin [66]. The cKO mouse displays a defective corneal innervation patterning consisting in abnormalities of the nerve projections similar to those described in a Semaphorin 3 deficient mouse models [78,79]. Semaphorin 3 regulates the navigation process of the nerves by guiding the direction of axon growth [79]. In our ciliary model, the decreased number of nerve bundles at E13.5 and the less centripetal migration of the corneal nerves at E18.5 suggest that the navigation process of trigeminal nerves in the cornea is affected.
Consistent with these exciting possibility, ciliogenic proteins including KIF3A, IFT88, and ARL13B, are required for the directional migration of GABAergic neurons [80,81]. Thus, assembly of the primary cilium on trigeminal nerves could be critical in restablishment of the corneal innervation following corneal transplant or during recovery of trauma of the cornea.
The Hh signal transduction pathway is intimately linked to the primary cilium [23,24], and its dosage is critical to ensure normal eye development [38,82,83]. In this study, we have shown

Histology, electron microscopy and immunofluorescence
Mouse embryos at different gestational stages were removed from Ift88 fl/fl females crossed with Wnt1-Cre;Ift88 fl/+ males and pictures were taken with a M80 stereomicroscope (Leica) equipped with a color IC80 HD camera (Leica). Half head or enucleated eyes from the embryos were fixed overnight in 4% paraformaldehyde (PFA) and embedded in paraffin for histological analysis. Hematoxylin and eosin (HE) staining was performed following standard procedures.
Corneal stromal thickness was determined in the center of the cornea on HE sections from 3 mice per age.
Endomucin and TUJ1 staining were performed on fixed whole corneas as previously described [85,86]. For immunofluorescence (IF) and X-gal staining on sections, half head and enucleated eyes from embryos were embedded and frozen in optimal cutting temperature compound (OCT Tissue-Tek, Sakura). For detection of the β-galactosidase activity by X-Gal staining, sections were fixed 10 min with 0.2% glutaraldehyde, 2 mM MgCl 2 in PBS at room temperature, and then incubated overnight in a solution containing 1 mg/mL Xgal, 5 mM K 3 Fe(CN), 5 mM K 4 Fe(CN), 0.01% deoxycholate, 0.02% NP40 and 2 mM MgCl 2 in PBS at 4°C. Sections were imaged with an AxioImager.Z2M microscope (Zeiss) equipped with an Axiocam 503 color camera (Zeiss).
For immunofluorescent staining, sections were fixed 10 min with 4% PFA at room temperature.
For transmission electron microscopy (TEM), embryonic eyes were processed as previously described [87]. Briefly, samples were fixed with 1% PFA and 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, post-fixed with 1% osmium tetroxide, embedded in Epon (Electron Microscopy Sciences) and stained with uranyl acetate and lead citrate after sectioning. Sections were imaged with Hitachi H7650 or S4300 microscopes.

SAG stimulation
Enucleated eyes from P0 mice were cut in half on the sagittal axis and the lens and the retina were removed. Eyeball pieces were incubated for 5h in keratinocyte-SFM with or without 100 nM SMO agonist (SAG, MilliporeSigma) at 37°C in 5% CO2. Tissues were fixed 12 min with 4% PFA in PBS, incubated 12 min with 5% Triton in PBS and then primary cilia and SMO were stained using a mouse anti-acetylated tubulin Ab (6-11B-1, Sigma-Aldrich) and a rabbit anti-SMO Ab (1:100, ABS1001 MilliporeSigma), respectively. Whole-mount eyeball tissues were imaged using a Zeiss LSM800 confocal. Cilia and SMO staining were also performed on ocular tissues from E14.5 embryos, without prior incubation with or without SAG.

Ex vivo imaging
Confocal imaging was performed on whole eyes from E18.5 Wnt1-Cre;Ift88 fl/fl ;R26 mT/mG and Wnt1-Cre;Ift88 fl/+ ;R26 mT/mG embryos as previously described [87]. Live GFP and tdTomato fluorescences were recorded by a Zeiss LSM880 confocal microscope, using a 40X water immersion objective lens and used to scan a 0.05 mm 2 field of view to study the keratocyte organization and 0.005 mm 2 field of view to quantify the cytoplasmic processes.  For visualization of the blood vessels at the corneal periphery, the microscope field was centered on the major arterial circle which was considered as the border between the cornea and the sclera (Supplement Figure 5). Serial optical sections were acquired from the epithelium to the presumptive iris (49 to 109 μm per z-stack). After maximum intensity projections, the corneal area covered by blood vessels and the percentage of corneal and scleral thicknesses with blood vessels were quantified with Fiji.

Intravital imaging
Intravital imaging of cilia in the mouse cornea was performed with an Olympus FV1200MPE microscope, equipped with a Chameleon Vision II Ti:Sapphire laser. Mice were anaesthetized with intraperitoneal injection of ketamine and xylazine (15 mg/mL and 1 mg/mL, respectively in PBS). A custom made stage was used to immobilize the mouse head and expose the eye globe. Mice were then placed under the microscope onto a heating pad and kept anesthetized with a continuous delivery of isoflurane through a nose cone (1% in air). A laser beam tuned at 930nm was focused through a 25X water immersion objective lens (XLPlan N, N.A.1.05; Olumpus USA) and used to scan a 0.5 mm 2 field of view. Serial optical sections were acquired in 2-3 μm steps, starting from the surface of the eye and capturing the entire thickness of the cornea (epithelium ~40 μm, stroma ~80 μm).  Table 1.

Statistical analysis
Data are presented as mean ± SEM. Student's t-tests were performed with Excel 2017 (Microsoft) and one-way ANOVA with post-hoc Tukey HSD test was performed with the online web statistical calculators https://astatsa.com/. A P value < 0.05 was considered significant.

AUTHOR CONTRIBUTIONS
C.P. and C.I. conceived and designed the study. C.P., P.L., P.R. and C.I. performed the experiments, analyzed and interpreted the data. C.I., P.L. and P.R. provided funding for the project. C.P. and C.I wrote the manuscript.

31.
Eibschitz-Tsimhoni, M.,   Boxed regions indicate the areas shown at higher magnification below. Keratocytes were counted in the stroma which is surrounded by red dotted lines. The keratocyte density is significantly increased in cKO embryos compared to control but not the total number of cells (n=4 embryos/group). Scale bar, 100 μm; ce, corneal epithelium. Data are presented as mean SEM. Statistical significance was assessed using two-tailed Student's t-test. ns, non-significant, P ≥ 0.05.  Fold change is expressed as mean ± SEM (n=3 embryos/group, both eyes of each embryo were pooled together). Only the expression of Hh pathway target genes was affected by the primary cilium ablation in the NCC. Statistical significance was assessed using two-tailed Student's t-test. ns, non-significant, P ≥ 0.05. (C) Hh activity assessed by Gli1-LacZ staining throughout the embryonic development. In the control embryos, the Hh activity progressively decreases in the POM from E12.5 to E18.5 but remains activated in a subpopulation of POM cells surrounding the RPE layer and extending until the iridocorneal angle, as well as in the POM surrounding the optic nerve (orange arrowheads). The primary cilium ablation in NCC leads to the absence of Hh activity in these specific areas. Hh signaling remains active in non-NCC derived tissues in the cKO embryos like in the RPE (white arrowheads) and the retina (asterisks). Scale bar, 100 μm.