Controlling the balance between direct and indirect neurogenesis in the developing cerebral cortex is mediated by cell-cell signaling (Cárdenas et al., 2018) and hence may involve the primary cilium. To investigate potential ciliary roles, we started characterizing cortical stem cell development in embryos mutant for the Inpp5e gene which has a prominent role in ciliary signaling and stability. Mutations in ciliary genes have previously been found to result in telencephalon patterning defects, most notably in a ventralization of the dorsal telencephalon and/or in defects at the corticoseptal (CSB) and pallial/subpallial boundaries (PSPB) (Ashique et al., 2009; Besse et al., 2011; Willaredt et al., 2008). Therefore, we first considered the possibility that such early patterning defects may be present in Inpp5e mutant embryos and could affect cortical stem cell development. In situ hybridization and immunofluorescence analyses of E12.5 control and Inpp5e^Δ/Δ embryos revealed no obvious effect on the expression of dorsal and ventral telencephalic markers at the corticoseptal boundary (Figure 1—figure supplement 1A–F). In contrast, the pallial/subpallial boundary was not well defined with a few scattered Pax6+ and Dlx2 expressing cells on the wrong side of the boundary, that is in the subpallium and pallium, respectively (Figure 1—figure supplement 1G–L). Moreover, the hippocampal anlage appeared smaller and disorganized with low level and diffuse expression of cortical hem markers (Figure 1—figure supplement 2), consistent with known roles of Wnt/β-catenin and Bmp signaling in hippocampal development (Galceran et al., 2000; Lee et al., 2000). In contrast, progenitors in the neocortical ventricular zone of Inpp5e mutant mice expressed the progenitor markers Emx1, Lhx2, Pax6 and Ngn2, though the levels of Pax6 protein expression appeared reduced in the medial neocortex suggestive of a steeper lateral to medial Pax6 expression gradient in mutant embryos. These expression patterns were maintained in E14.5 Inpp5e^Δ/Δ embryos but revealed an area in the very caudal/dorsal telencephalon where the neocortex was folded (Figure 1—figure supplement 3). These folds became more prominent at more caudal levels and were also present in the hippocampal anlage. Taken together, these findings indicate that Inpp5e mutants have mild patterning defects affecting the integrity of the PSPB, hippocampal development and the caudal-most neocortex while the rostral neocortex shows no gross malformation or mispatterning and can therefore be analyzed for effects of the Inpp5e mutation on direct and indirect neurogenesis.

Based upon these findings, we started analyzing the proliferation and differentiation of RGCs in Inpp5e^Δ/Δ embryos in the rostrolateral and rostromedial neocortex to avoid the regionalization defects described above. As a first step, we determined the proportion of RGCs, basal progenitors and neurons in these regions in E12.5 embryos. Double immunofluorescence for PCNA which labels all progenitor cells (Hall et al., 1990) and the radial glial marker Pax6 did not reveal differences in the proportions of RGCs at both medial and lateral levels (Figure 1A–D). In contrast, the proportion of Tbr2+ basal progenitors was reduced laterally but not medially (Figure 1E–H). This decrease coincided with an increase in Tbr1+ neurons specifically in the lateral neocortex (Figure 1I–L).

To determine whether these alterations are maintained at a later developmental stage, we repeated this investigation in E14.5 embryos. This analysis revealed no significant differences in the proportion of Pax6+ RGCs (Figure 2A–D). Similarly, there was no alteration in the proportion of Tbr2+ basal progenitors in lateral neocortex; however, their proportion was reduced medially (Figure 2E–H). To label cortical projection neurons, we used double immunofluorescence for Tbr1 and Ctip2 which allowed us to distinguish between Tbr1+Ctip2+ and Tbr1-Ctip2+ neurons. Quantifying these subpopulations showed no effect on the formation of Tbr1+ Ctip2+ neurons in Inpp5e^Δ/Δ embryos. In contrast, the proportion of Tbr1- Ctip2+ neurons was reduced medially but increased in the lateral neocortex (Figure 2I–N). Taken together with our E12.5 analyses, these findings show that in the lateral neocortex of Inpp5e^Δ/Δ an increase in the proportion of Tbr1+ neurons at E12.5 is followed by an augmented fraction of Tbr1- Ctip2+ neurons at E14.5, whereas the proportion of basal progenitors recovered after an initial down-regulation.

To address the defective cellular processes underlying these neurogenesis defects in Inpp5e mutants, we first investigated programmed cell death and found few apoptotic cells in the control and mutant cortex (Figure 3—figure supplement 1). Next, we measured proliferation rates of cortical progenitors and performed double immunofluorescence for PCNA and pHH3 which labels mitotic RGCs located at the ventricular surface and dividing basal progenitors in abventricular positions (Figure 3—figure supplement 2). This analysis revealed no statistically significant differences in the E12.5 and E14.5 lateral neocortex of control and Inpp5e^Δ/Δ embryos. The proportion of mitotic apical and basal progenitors, however, was reduced in the E12.5 medial neocortex (Figure 3—figure supplement 2). Interestingly, this decrease in the fraction of mitotic basal progenitors precedes the reduced proportion of basal progenitors in the E14.5 medial neocortex (Figure 2E–H).

The cell cycle represents another key regulator of neuronal differentiation and a mutation in Kif3a affects ciliogenesis and the cell cycle in the developing neocortex (Wilson et al., 2012). To investigate the possibility of altered cell cycle kinetics, we used a BrdU/IdU double labeling strategy (Martynoga et al., 2005; Nowakowski et al., 1989) to determine S phase length and total cell cycle length in RGCs and found no statistically significant changes in these parameters (Figure 3—figure supplement 3).

Finally, the increased neuron production could also be explained by an increase in direct neurogenesis at the expense of basal progenitor cell formation. To test this possibility, we gave BrdU to E11.5 pregnant mice 24 hr before dissecting the embryos. We then used BrdU immunostaining in conjunction with Tbr1 and Tbr2 to identify the neurons and basal progenitors formed in the lateral neocortex within the 24 hr time period. This analysis showed that the proportion of Tbr1+ neurons compared to the total number of BrdU+ cells increased while the Tbr2+ proportion decreased in Inpp5e mutants (Figure 3). Since the cell cycle of basal progenitors is longer than 24 hr (Arai et al., 2011), the 24 hr interval used in our cell cycle exit experiment was too short for newly formed basal progenitors to undergo one additional round of the cell cycle and as the BrdU label would have been diluted with a further round of division, this analysis supports our hypothesis that direct neurogenesis became more prevalent in Inpp5e^Δ/ΔRGCs.

Next, we investigated the consequences of this increase in direct neurogenesis on cortical size and layer formation. Since Inpp5e^Δ/Δ newborn pups die perinatally (Bielas et al., 2009), we focused our analysis on E18.5 embryos. The mutant lacked obvious olfactory bulbs, as revealed by whole mounts of control and mutant brains (Figure 4—figure supplement 1). To gain insights into the overall histology of the mutant forebrain, we stained coronal sections with DAPI. This analysis showed that most of the mutant cortex was thinner except for the rostrolateral level (Figure 4—figure supplement 2). In addition, the hippocampus was malformed with a smaller dentate gyrus. Investigating the expression of markers characteristic of the entire hippocampus (Nrp2; Galceran et al., 2000), the CA1 field (Scip1; Frantz et al., 1994) and the dentate gyrus (Prox1; Oliver et al., 1993) showed that these hippocampal structures were present but were severely reduced in size and disorganized in Inpp5e^Δ/Δ embryos (Figure 4—figure supplement 3). In addition, the corpus callosum, the major axon tract connecting the two cerebral hemispheres, was smaller. We confirmed this effect by staining callosal axons and surrounding glial cells that guide these axons to the contralateral hemisphere with L1 and GFAP, respectively (Figure 4—figure supplement 4).

After characterizing the gross morphology of the Inpp5e^Δ/Δ cortex, we next investigated whether the increased neuron formation in E12.5 mutant embryos led to changes in the neuronal subtype composition of the E18.5 cortex. To this end, we used immunofluorescence labeling for Tbr1 and Ctip2 to analyze the formation of layer VI and V neurons, respectively, whereas Satb2 served as a layer II-IV marker (Figure 4). Inspecting these immunostainings at low magnification showed that Tbr1+, Ctip2+ and Satb2+ neurons occupied their correct relative laminar positions in Inpp5e mutants (Figure 4A–F) except for neuronal heterotopias which were present in all mutant brains, although their number and position varied (Figure 4D). These immunostainings also revealed a medial shift in the position of the rhinal fissure, a sulcus that is conserved across mammalian species and separates neocortex from the paleocortical piriform cortex (Ariens-Kapers et al., 1936). This shift was more marked caudally and suggests a dramatic expansion of the Inpp5e mutant piriform cortex at the expense of neocortex at caudal most levels (Figure 4D–F). Using the Tbr1/Ctip2 and Satb2 stainings, we determined the proportions of deep and superficial layer neurons, respectively. Because of the expanded piriform cortex in Inpp5e mutants, we limited this investigation to the unaffected rostral neocortex. In the rostrolateral neocortex, we found the proportion of Tbr1+ neurons to be reduced (Figure 4G,H,M). This reduction coincided with an increased proportion of Ctip2+ layer V neurons (Figure 4I,J,N) while the Satb2 population was unchanged (Figure 4K,L,O). In contrast, the rostromedial neocortex did not show any differences (Figure 4P–X). Thus, the increase in direct neurogenesis in the lateral neocortex during earlier development concurs with a change in the proportions of E18.5 Tbr1+ and Ctip2+ deep layer neurons.

To start to unravel the mechanisms by which Inpp5e controls cortical stem cell development, we first analyzed whether the increased early neurogenesis is restricted to Inpp5e^Δ/Δ mutants or is observed in another mutant affecting cilia. To this end, we focused on the Tectonic 2 (TCTN2) gene which is crucial for ciliary transition zone architecture (Shi et al., 2017) and which, like INPP5E, is mutated in Joubert Syndrome (Garcia-Gonzalo et al., 2011). Interestingly, E12.5 Tctn2^Δ/Δ mutant embryos (Reiter and Skarnes, 2006) also showed an increased proportion of Tbr1+ projection neurons and a concomitant decrease in Tbr2+ basal progenitors in the dorsolateral telencephalon (Figure 5). Due to embryonic lethality, however, we were not able to investigate the formation of cortical neurons at later stages.

Our findings in the Inpp5e and Tctn2 mutants suggested a role for cilia in cortical progenitor cells to control early neurogenesis. Therefore, we examined the presence and the structure of primary cilia in the developing forebrain of Inpp5e^Δ/Δ embryos by immunofluorescence and electron microscopy. We first analyzed the presence of the small GTPase Arl13b, enriched in ciliary membranes, and of γ-Tubulin (γTub), a component of basal bodies (Caspary et al., 2007). We found no major difference in the number or the apical localization of cilia in control and Inpp5e^Δ/Δ neuroepithelial cells in the E12.5 telencephalon (Figure 6A,B) or diencephalon (data not shown).

To gain insights into the fine structure of these primary cilia, we performed electron microscopy analyses. Scanning electron microscopy (SEM) provided an observation of the cilia protruding into the telencephalic ventricles. In control embryos, almost all RGCs had a single, ~1 μm long primary cilium (Figure 6C), as previously described (Besse et al., 2011). Some Inpp5e^Δ/Δ mutant cells also displayed an apparently normal cilium (Figure 6D,E), whereas other cells harbored abnormal cilia, either with a lateral blob (arrowhead in Figure 6D,E) or as a short and bloated cilium-like protrusion (arrows in Figure 6D,E).

Transmission electron microscopy (TEM) confirmed the presence of abnormal cilia in Inpp5e^Δ/Δ embryos. Cilia were recognized by basal bodies anchored to the apical membrane in both control and Inpp5e^Δ/ΔRGCs (Figure 6F–L,N,Q). However, in Inpp5e^Δ/Δ cells, some cilia lacked the axoneme and showed unusual membranous structures that resemble budding vesicles emerging from the lateral surface of the cilium (Figure 6G,K), internal vesicles (arrows in Figure 6I,K,L), or undulating peripheral membranes (Figure 6I), indicating an Inpp5e-dependent defect in ciliary membrane morphology. Transverse sections revealed the presence of cilia with apparently normal 9+0 axonemes, as well as cilia containing abnormal numbers of microtubule doublets in Inpp5e^Δ/Δ embryos (Figure 6O,P). To quantify these ciliary defects, we counted the number of normal versus abnormal cilia on TEM images obtained from control and Inpp5e^Δ/Δ embryos, and found an increase in abnormal cilia in Inpp5e^Δ/Δ compared to control embryos (Figure 6R). Taken together, a significant number of abnormal primary cilia were found at the apical end of E12.5 RGCs in the forebrain of Inpp5e^Δ/Δ embryos. These abnormalities are consistent with a role of Inpp5e in maintaining cilia stability (Jacoby et al., 2009).

Primary cilia play a crucial role in Shh signaling by controlling the proteolytic cleavage of full-length Gli3 (Gli3FL) into the Gli3 repressor form (Gli3R) in the absence of Shh and by converting Gli3FL into the transcriptional activator Gli3A in the presence of Shh. Moreover, the dorsal telencephalon predominately forms Gli3R (Fotaki et al., 2006) and mice that can only produce Gli3R have no obvious defect in cortical development (Besse et al., 2011; Bose, 2002). In addition, we recently showed that Gli3 has a prominent role in RGCs controlling the switch from symmetric proliferative to asymmetric neurogenic cell division (Hasenpusch-Theil et al., 2018). Therefore, we hypothesized that alterations in Gli3 processing caused by abnormal cilia function underlies the increased direct neurogenesis and the cortical malformations in Inpp5e^Δ/Δ embryos. In situ hybridization showed that Gli3 mRNA expression might be slightly reduced but the overall expression pattern in the telencephalon remains unaffected (Figure 7—figure supplement 1). We next investigated the formation of Gli3FL and Gli3R in the E12.5 dorsal telencephalon of Inpp5e^Δ/Δ embryos using western blots. This analysis revealed no change in the levels of Gli3FL but a significant decrease inGli3R which resulted in a reduced Gli3R to Gli3FL ratio in the mutant (Figure 7A–D) suggesting that the Inpp5e mutation affects Gli3 processing.

The next set of experiments aimed to clarify a role for the reduced Gli3 processing. To this end, we restored Gli3R levels by crossing Inpp5e mutants with Gli3^Δ699/+ mice that can only produce Gli3R in a cilia-independent manner (Besse et al., 2011; Bose, 2002). Overall inspection of Inpp5e^Δ/Δ;Gli3^Δ699/+ embryos revealed restored eye formation, whereas Inpp5e^Δ/Δ embryos either completely lacked eyes or showed microphthalmia (Jacoby et al., 2009; Figure 7—figure supplement 2). Moreover, the overall morphology of the telencephalon is much improved in Inpp5e^Δ/Δ;Gli3^Δ699/+ embryos as compared to Inpp5e^Δ/Δ embryos. In E18.5 Inpp5e^Δ/ΔGli3^Δ699/+ mutants, the corpus callosum has a thickness indistinguishable from that of control embryos (Figure 7—figure supplement 3). In E12.5 and E14.5 Inpp5e^Δ/Δ;Gli3^Δ699/+ embryos, the neocortex lacks the undulations of the VZ present in Inpp5e^Δ/Δ embryos (data not shown) and the morphology of the hippocampal anlage is more akin to that in wild-type embryos but it is still smaller and less bulged (Figure 7E,G,I).

We also determined the proportions of basal progenitors and Tbr1+ neurons at E12.5 which were decreased and increased, respectively, in the lateral neocortex of Inpp5e^Δ/Δ embryos. While these changes were still present in Inpp5e^Δ/Δ littermate embryos, there was no statistically significant difference between control and Inpp5e^Δ/Δ;Gli3^Δ699/+ embryos (Figure 7E–L). This finding indicates that the neurogenesis phenotype of E12.5 Inpp5e^Δ/Δ mutants is rescued by a single copy of the Gli3^Δ699 allele. We next investigated the formation of basal progenitors and of cortical projection neurons in E14.5 Inpp5e^Δ/Δ;Gli3^Δ699/+ embryos. The proportion of Tbr1+Ctip2+ neurons was not affected in the medial neocortex of E14.5 Inpp5e^Δ/Δ;Gli3^Δ699/+ embryos. In contrast, the proportion of Tbr1-Ctip2+ neurons was reduced as in Inpp5e^Δ/Δ mutants (Figure 8A,C,E,I,J). Similarly, the proportions of basal progenitors in the medial Inpp5e^Δ/ΔGli3^Δ699/+ neocortex was slightly improved compared to Inpp5e^Δ/Δ embryos but significantly smaller than in control embryos (Figure 8B,D,F,K). As re-introducing a single Gli3^Δ699 allele does not completely rescue the Inpp5e^Δ/Δ neurogenesis phenotype, we generated Inpp5e^Δ/Δ embryos homozygous for the Gli3^Δ699 allele. Interestingly, the morphology of the dorsal telencephalon including the hippocampal formation was indistinguishable between control and Inpp5e^Δ/Δ;Gli3^Δ699/Δ699 embryos (Figure 8A,B,G,H) and the formation of Tbr1-Ctip2+ neurons and Tbr2+ basal progenitors were not affected (Figure 8G,H,J,K). Taken together, these findings indicate that re-introducing a single copy of the Gli3R allele into the Inpp5e mutant background leads to a partial rescue of cortical neurogenesis in Inpp5e^Δ/Δ embryos, whereas two copies are required for a full rescue.

RGCs in the developing mouse neocortex have the potential to undergo symmetric proliferative or asymmetric cell divisions with the latter division mode producing neurons in a direct manner or indirectly via basal progenitors. Balancing out these division modes is important not only to determine final neuronal output and cortical size but also the types of cortical projection neurons and, hence, subtype composition of the adult neocortex. In the E12.5 Inpp5e and Tctn2 mouse mutants, we identified an increased formation of neurons in the lateral neocortex. Based on our cell cycle exit experiment, additional neurons are formed from RGCs at the expense of basal progenitors. Given the cell cycle length of basal progenitors of >24 hr (Arai et al., 2011), it is unlikely that new born basal progenitors would have undergone an additional round of cell division to produce two neurons within the time frame of this experiment. Such an extra division would also have diluted the BrdU label. We therefore conclude that the Inpp5e mutation caused RGCs to preferentially produce neurons directly. Moreover, neurogenesis defects only became obvious at E14.5 in the medial neocortex. This delay might reflect the neurogenic gradient in the neocortex or might be related to specific gene expression changes such as reduced Pax6 expression in medial neocortical progenitors.

Interestingly, the increase in direct neurogenesis led to an increased proportion of Ctip2+ deep layer V neurons in the E18.5 neocortex but did not coincide with a reduced proportion of upper layer neurons. This effect could be explained in several mutually non-exclusive ways. First, neurons born at E12.5 initially express both Ctip2 and Tbr1 (Figure 7) and later down-regulate Ctip2. Inpp5e could therefore affect the signaling that controls this downregulation. Secondly, the proportions of basal progenitors and neurons were normalized in E14.5 mutants. Since basal progenitors are a main source of upper layer neurons (Arnold et al., 2008; Vasistha et al., 2015), this normalization would account for the sufficient numbers of Satb2+ upper layer neurons. Newly formed projection neurons signal back to RGCs via Jag1, Fgf9 and Neurotrophin 3 (Parthasarathy et al., 2014; Seuntjens et al., 2009; Wang et al., 2016) to control the sequential production of deep and upper layer neurons and of glia (Silva et al., 2019). Inpp5e might affect these signals by controlling cilia stability and/or levels of PI(3,4,5)P₃ (Bielas et al., 2009; Jacoby et al., 2009) that acts as a second messenger in receptor tyrosine kinase signaling. Regardless of the exact mechanism, our findings suggest a novel, spatially and temporally restricted role for Inpp5e in controlling the decision between direct and indirect neurogenesis. This function differs from those described for other cilia mutants. Conditional inactivation of Ift88 and Kif3a leads to a larger cortex (Foerster et al., 2017; Wilson et al., 2012) with a modest increase in BP production in the absence of a delay in neurogenesis (Foerster et al., 2017) while Rpgrip1l mutants have reduced numbers of both basal progenitors and neurons (Postel et al., 2019). These findings highlight the multiple and varied roles cilia play in cortical development.

Our study also shed lights into the mechanisms by which Inpp5e controls the decision between direct and indirect neurogenesis. Most notably, the Gli3R level and Gli3R/Gli3FL ratio are decreased in Inpp5e^Δ/Δ embryos. While the Inpp5e mutation does not lead to an up-regulation of Shh signaling in the dorsal telencephalon (Magnani et al., 2015), re-introducing a single or two copies of Gli3R in an Inpp5e mutant background partially and fully restores the neurogenesis defects, respectively. This rescue indicates that reduced levels of Gli3R rather than the reduction in the Gli3R/Gli3FL ratio are responsible for the prevalence of direct neurogenesis in Inpp5e^Δ/Δ embryos. This idea is consistent with the findings that (i) Gli3^Δ699/Δ699 embryos that cannot produce Gli3FL and Gli3A show no obvious phenotype in cortical development (Besse et al., 2011; Bose, 2002), (ii) dorsal telencephalic patterning defects in Gli3^Xt/Xt mutants are not rescued in Shh^-/-/Gli3^XtXt double mutants (Rallu et al., 2002; Rash and Grove, 2007), (iii) Shh promotes the generation of olfactory bulb interneurons and cortical oligodendrocytes and neurogenesis in the subventricular zone by reducing Gli3R rather than by promoting Gli activator function (Petrova et al., 2013; Wang et al., 2014; Zhang et al., 2020). In addition, there is also a dramatic rescue of eye development and the rescue also extends to other malformations of the Inpp5e^Δ/Δ forebrain, including the corpus callosum, the hippocampus and the expansion of the piriform cortex, structures that are also affected in Gli3 null and hypomorphic mutants (Amaniti et al., 2015; Johnson, 1967; Magnani et al., 2014; Theil et al., 1999; Wiegering et al., 2019). Taken together, these findings support the idea that Inpp5e and the primary cilium control key processes in cortical development by regulating the formation of Gli3R.

Our analyses support several mutually non-exclusive mechanisms how the Inpp5e mutation impacts on Gli3 processing. First, our electron microscopy study revealed severe structural abnormalities in large proportions of cilia. The Inpp5e phosphatase hydrolyses PI(3,4,5)P₃, which is essential for the effective activation of the serine threonine kinase Akt (Kisseleva et al., 2002; Plotnikova et al., 2015). Following PI(3,4,5)P₃ binding, Akt translocates to the membrane and becomes phosphorylated at T308 by phosphoinositide-dependent kinase-1 (Pdk1) and at S473 by mammalian target of rapamycin complex (mTORC2) (Yu and Cui, 2016). Consistent with the loss of Inpp5e function and a resulting increase in PI(3,4,5)P₃, western blot analysis revealed elevated pAkt^S473 levels (data not shown). Increased phosphorylation at this site has been implicated in inhibiting cilia assembly and promoting cilia disassembly (Mao et al., 2019) and could hence explain the structural defects of RGC Inpp5e^Δ/Δ cilia. Secondly, Inpp5e could control Gli3 processing through its effect on the transition zone (TZ). It is required for TZ molecular organization (Dyson et al., 2017) and its substrate PI(4,5)P2 plays a role in TZ maturation in Drosophila (Gupta et al., 2018). This model is further supported by our finding that a mouse mutant for the TZ protein Tctn2 phenocopies the Inpp5e^Δ/Δ neurogenesis defect. In turn, several mouse mutants defective for TZ proteins are required for Inpp5e localization to cilia and show microphthalmia (Garcia-Gonzalo et al., 2011; Garcia-Gonzalo et al., 2015; Sang et al., 2011; Yee et al., 2015). Tctn proteins are also required for Gli3 processing (Garcia-Gonzalo et al., 2011; Sang et al., 2011; Thomas et al., 2012; Wang et al., 2017) and the TZ protein Rpgrip1l controls the activity of the proteasome at the basal body responsible for proteolytic cleavage of Gli3 (Gerhardt et al., 2015). Taken together, these findings indicate that Inpp5e mutation might affect the ability of RGCs to switch to indirect neurogenesis through defects in cilia stability and/or the integrity of the ciliary transition zone (Figure 9).

In humans, hypomorphic INPP5E mutations contribute to Joubert Syndrome (JS), a ciliopathy characterized by cerebellar malformations and concomitant ataxia and breathing abnormalities. In addition, a subset of JS patients exhibit cortical abnormalities including polymicrogyria, neuronal heterotopias and agenesis of the corpus callosum (Poretti et al., 2011). Strikingly, the Inpp5e mouse mutant also shows several of these abnormalities. In the caudal telencephalon, the otherwise lissencephalic cortex formed folds reminiscent of the polymicrogyria in JS patients. In addition, the mutant formed leptomeningeal heterotopias with 100% penetrance, but their number and location varied. Mutations in ciliary genes were previously associated with heterotopia formation in humans and mice (Magnani et al., 2015; Uzquiano et al., 2019). Mice carrying mutations in the Eml1 gene encoding a microtubule-associated protein show subcortical heterotopias due to a mispositioning of RGCs and impaired primary cilia formation (Uzquiano et al., 2019). Finally, the corpus callosum is thinner but callosal axons project to the contralateral cerebral hemisphere in Inpp5e mutants. This phenotype is milder compared to that of other mouse mutants with altered cilia that show complete agenesis of the corpus callosum with callosal axons forming Probst bundles (Benadiba et al., 2012; Laclef et al., 2015; Putoux et al., 2019). Unlike these other ciliary mutants, the corticoseptal boundary which plays a crucial role in positioning guidepost cells that control midline crossing of callosal axons (Magnani et al., 2014) is not obviously affected in Inpp5e^Δ/Δ embryos. Instead, the thinner corpus callosum is likely to be the result of reduced size of the caudal neocortex. Despite these differences, however, re-introducing Gli3R into the cilia mutant background restores callosal development in both groups of mutants suggesting that cilia control two independent steps in corpus callosum formation by regulating Gli3 processing. Thus, the Inpp5e^Δ/Δ mutant recapitulates cortical abnormalities in JS patients and starts to help unravelling the pathomechanisms underlying these defects.

All experimental work was carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and UK Home Office guidelines. All protocols were reviewed and approved by the named veterinary surgeons of the College of Medicine and Veterinary Medicine, the University of Edinburgh, prior to the commencement of experimental work. Inpp5e^Δ (Inpp5e^delta), Gli3^Δ699 (Gli3^delta699) and Tctn2^Δ (Tctn2^tm1.1Reit) mouse lines have been described previously (Bose, 2002; Garcia-Gonzalo et al., 2011; Jacoby et al., 2009). Inpp5e^Δ/+ mice were interbred to generate Inpp5e^Δ/Δ embryos; exencephalic Inpp5e^Δ/Δ embryos which made up ca. 25% of homozygous mutant embryos were excluded from the analyses. Wild-type and Inpp5e^Δ/+ litter mate embryos served as controls. Inpp5e^Δ/Δ;Gli3^Δ699/+ and Inpp5e^Δ/Δ;Gli3^Δ699/Δ699 embryos were obtained from inter-crosses of Inpp5e^Δ/+;Gli3^Δ699/+ mice using wild-type, Inpp5e^Δ/+ and Gli3^Δ699/+ embryos as controls. Embryonic (E) day 0.5 was assumed to start at midday of the day of vaginal plug discovery. Transgenic animals and embryos from both sexes were genotyped as described (Bose, 2002; Jacoby et al., 2009). For each marker and each stage, three to eight embryos were analyzed.

For measuring cell cycle lengths, pregnant females were intraperitoneally injected with a single dose of IdU (Sigma-Aldrich) (10mg/ml) at E12.5, followed by an injection of BrdU (Sigma-Aldrich) (10 mg/ml) 90 min later. Embryos were harvested 30 min after the second injection. For cell cycle exit analyses, BrdU was injected peritoneally into E11.5 pregnant females and embryos were harvested 24 hr later.

For immunohistochemistry, embryos were fixed overnight in 4% paraformaldehyde, incubated in 30% sucrose at +4°C for 24 hr, embedded in 30% sucrose/OCT mixture (1:1) and frozen on dry ice. Immunofluorescence staining was performed on 12 to 14 μm cryostat sections as described previously (Theil, 2005) with antibodies against Arl13b (mouse) (Neuromab 75–287; 1:1500), rat anti-BrdU (1:50, Abcam #ab6326), mouse anti-BrdU/IdU (B44) (1:50, BD Biosciences #347580), rabbit anti-Cleaved Caspase 3 (1:100, Cell Signaling Technology, #9664), rat anti-Ctip2 (1:1000, Abcam #ab18465), rabbit anti-GFAP (1:1000, Agilent/Dako #Z 0334), rat anti-L1, clone 324 (1:1000, Millipore #MAB5272), rabbit anti-Pax6 (1:400, Biolegend #901301), mouse anti-PCNA (1:500, Abcam #ab29), rabbit anti-Prox1 (1:1000, RELIATech #102-PA32). rabbit anti-pHH3 (1:100, Millipore #06–570), mouse anti-Satb2 (1:200, Abcam #ab51502), rabbit anti-Tbr1 (1:400, Abcam #ab31940), rabbit anti-Tbr2 (1:1000, Abcam #ab23345) and rabbit anti-γTUB (Sigma-Aldrich SAB4503045; 1:100). Primary antibodies for immunohistochemistry were detected with Alexa- or Cy2/3-conjugated fluorescent secondary antibodies. The Cleaved Caspase three and Tbr1 signals were amplified using biotinylated secondary IgG antibody (swine anti-rabbit IgG) (1:400, Dako) followed by Alexa Fluor 488 (1:100, Invitrogen) or 568 Streptavidin (1:100, Thermo Fisher Scientific). For counter staining DAPI (1:2000, Thermo Fisher Scientific) was used. Prox1 protein was detected non-fluorescently using biotinylated goat anti-rabbit IgG (1: 400,Agilent (Dako)) followed by avidin-HRP and DAB detection using Vectastain Elite ABC peroxidase kit (Vector laboratories) as described previously (Magnani et al., 2010).

In situ hybridization on 12 μm serial paraffin sections were performed as described previously (Theil, 2005) using antisense RNA probes for Axin2 (Lustig et al., 2002), Bmp4 (Jones et al., 1991), Dbx1 (Yun et al., 2001), Dlx2 (Bulfone et al., 1993), Emx1 (Simeone et al., 1992), Gli3 (Hui et al., 1994), Lhx2 (Liem et al., 1997), Msx1 (Hill et al., 1989), Ngn2 (Gradwohl et al., 1996), Nrp2 (Galceran et al., 2000), Pax6 (Walther and Gruss, 1991), Scip1 (Frantz et al., 1994), Wnt2b (Grove et al., 1998).

Protein was extracted from the dorsal telencephalon of E12.5 wild-type and Inpp5e^Δ/Δ embryos (n = 4 samples per genotype) as described previously (Magnani et al., 2010). 10 μg protein lysates were subjected to gel electrophoresis on a 3–8% NuPAGE Tris-Acetate gel (Life Technologies), and protein was transferred to a Immobilon-FL membrane (Millipore), which was incubated with goat anti-h/m Gli3 (1:500, R and D Systems #AF3690) and mouse anti-β-Actin antibody (1:15000, Abcam #ab6276). After incubating with donkey anti-goat IgG IRDye680RD (1:15000, LI-COR Biosciences) and donkey anti-mouse IgG IRDye800CW secondary antibodies (1:15000, Life Technologies), signal was detected using LI-COR’s Odyssey Infrared Imaging System with Odyssey Software. Values for protein signal intensity were obtained using Image Studio Lite Version4.0. Gli3 repressor and full-length protein levels and the Gli3 repressor/full length were compared between wild-type and mutant tissue using an unpaired t-test.

TEM and SEM image acquisition were performed in the Cochin Imaging Facility and on the IBPS EM Facility, respectively. For scanning electron microscopy, embryos were dissected in 1.22x PBS (pH 7.4) and fixed overnight with 2% glutaraldehyde in 0.61x PBS (pH 7.4) at 4°C. Heads were then sectioned to separate the dorsal and ventral parts of the telencephalon, exposing their ventricular surfaces. Head samples were washed several times in 1.22x PBS and postfixed for 15 min in 1.22x PBS containing 1% OsO4. Fixed samples were washed several times in ultrapure water, dehydrated with a graded series of ethanol and prepared for scanning electron microscopy using the critical point procedure (CPD7501, Polaron). Their surfaces were coated with a 20 nm gold layer using a gold spattering device (Scancoat Six, Edwards). Samples were observed under a Cambridge S260 scanning electron microscope at 10 keV.

For transmission electron microscopy tissues were fixed for 1 hr with 3% glutaraldehyde, post-fixed in 1.22x PBS containing 1% OsO4, then dehydrated with a graded ethanol series. After 10 min in a 1:2 mixture of propane:epoxy resin, tissues were embedded in gelatin capsules with freshly prepared epoxy resin and polymerized at 60°C for 24 hr. Sections (80 nm) obtained using an ultramicrotome (Reichert Ultracut S) were stained with uranyl acetate and Reynold’s lead citrate and observed with a Philips CM10 transmission electron microscope.

Data were analyzed using GraphPadPrism eight software with n = 3–8 embryos for all analyses. Shapiro-Wilk normality tests informed whether to use t-tests for normally distributed data and Mann Whitney tests for data which did not pass the normality test. Cortical thickness was analyzed using a two-way ANOVA followed by Sidak’s multiple comparisons test. A fisher’s exact test was used to analyze the quantification of normal and abnormal cilia. The Gli3 rescue experiments were evaluated with one way ANOVAS followed by Tukey’s multiple comparisons test. A single asterisk indicates significance of p<0.05, two asterisks indicate significance of p<0.01 and three asterisks of p<0.001. Due to morphological changes blinding was not possible and scores were validated by a second independent observer. Supplementary file 1-table 1 provides a summary of test statistics.