Introduction

The primary cilium is a tiny microtubule-based organelle present on the surface of nearly all mammalian cell types including neurons, which functions as a signaling hub and transduces several signaling pathways comprising Sonic Hedghog (SHH), Wnt, Delta/Notch and mTOR pathways ¹. Primary cilium dysfunction causes pleiotropic diseases named ciliopathies. KIF7 is a ciliary kinesin responsible for the trafficking and the positive and negative regulation of the GLI transcription factors in the primary cilium of mammals ². Loss of function of KIF7 in the mouse has shown that KIF7 regulates (SHH) signalling by acting downstream of Smoothened (Smo) and upstream of GLI2 and GLI3 ^3–7. Studies in mice established that KIF7 activity is dependent on the expression level of SHH. In the absence of SHH, KIF7 localizes to the base of the primary cilium ⁵. GLI factors are phosphorylated and addressed to the proteasome at the base of the primary cilium for degradation, leading to the formation of a cleaved and stable transcriptional repressor (GLI3-R). In the presence of SHH, KIF7 accumulates at the distal tip of the primary cilium ^4,5 and associates with full length GLI2/3 that become transcriptional activators (GLI-A) ⁸.

Since 2011, ten studies have identified patients carrying mutations in the KIF7 gene responsible of ciliopathies classified according to clinical features as hydrolethalus, acrocallosal, Joubert and Greig cephalopolysyndactyly syndromes ^9–18. MRI investigations revealed macrocephaly, ventricles enlargement and corpus callosum alterations. They also showed the characteristic hindbrain abnormalities observed in most ciliopathies such as molar tooth sign (MTS) and cerebellar atrophy. Patients also presented with mild but frequent cortical malformations as well as neurodevelopmental delay, intellectual disability and seizures ^10,13,16,19 which indicate cortical abnormalities.

In physiological conditions, the proper activity of the cortex relies on the excitatory/inhibitory balance i.e. on the ratio, positioning and connectivity of excitatory glutamatergic (principal neurons, PN) and inhibitory GABAergic interneurons (cIN), generated from progenitors in the dorsal and ventral telencephalon, respectively. The cortical neurons of dorsal and ventral origin each migrate towards the developing cortex, the so-called cortical plate (CP), according to a specific timeline allowing the establishment of proper connections in the CP.

SHH plays a central role in establishing the dorso-ventral patterning of the forebrain, and in regulating the proliferation and differentiation of cortical progenitors of dorsal and ventral origins. At early embryonic stage, the ventral expression of SHH orchestrates the ventro-dorsal ^20,21 and medio-lateral ²² regionalization of the mouse forebrain and the differentiation of ventral cell types ²⁰. Shh ablation performed after forebrain patterning alters the specification of distinct subgroups of GABAergic interneurons ^23–26. Interestingly, conditional ablation of Shh and Smo in the embryonic cortex reduces the proliferation of dorsal progenitors ²⁷, demonstrating a minimal SHH expression in the dorsal telencephalon, even before birth ²⁸. Beside its role on proliferation and specification, SHH also controls the migration of interneurons to the cortical plate ^29–33. In the embryonic telencephalon, GLI transcription factors mediate SHH signals in complex and specific ways. The three GLI factors identified in mammals are expressed in the mouse forebrain: GLI1 along the source of SHH in the ganglionic sulcus, and GLI2 and GLI3 dorsally to the SHH source ^26,34,35. GLI1 acts uniquely as a pathway activator whereas GLI2 and GLI3 can be processed in transcriptional activators or inhibitors in the primary cilium. However, both GLI1 and GLI2 function primarily as transcriptional activators in response to SHH activity in the ventral forebrain ³⁵. Nevertheless, GLI1 and GLI2 mutants show mild phenotypes ^36,37 and GLI2 seems required to transduce high level SHH signals in mice ³⁸. In contrast, the development of the dorsal cortex, where principal excitatory neurons differentiate, depends mainly on the expression of the GLI3-R repressor, revealing low cortical expression of SHH ^39,40. Following patterning, Gli3R/A ratio remains critical for specifying the fate of cortical progenitors and regulating cell cycle kinetics ^41,42.

Previous studies investigated the mechanisms underlying the corpus callosum agenesis in patients with KIF7 mutation using Kif7 knock-out (Kif7 -/-) mice ⁴³ and the consequence of KIF7 knock down on cortical neurogenesis in principal cells electroporated with Kif7 shRNA⁴⁴. Nevertheless, the influence of developmental abnormalities associated with ciliopathies on the cortical cytoarchitecture is poorly understood. Given the crucial role of KIF7 in regulating the GLI pathways and the important role of Gli3-R and GLI2/3-A in regulating the early developmental stages of the dorsal and ventral telencephalon, respectively, we examined here the cortical development, the establishment of long distance projections with the thalamus and the migration of GABAergic interneurons (cIN) in Kif7 -/- mice. We focused at embryonic stage E14.5, a key period in corticogenesis when the cortical plate begins to form and cIN start to invade the developing cortex. The influence of abnormal SHH signaling in Kif7 -/- mice on the migratory behavior of cIN was investigated by comparing the migration of cIN in living Kif7 -/- cortical slices and in control slices treated with pharmacological activator and inhibitor of the SHH pathway. We moreover determined the local distribution of the SHH protein in the embryonic cortex.

Our results show developmental defects leading to permanently displaced neurons, abnormal formation of cortical layers and defective cortical circuits that could be responsible for epilepsy and/or intellectual deficiency in patients carrying KIF7 mutation ^9–18.

Results

Kif7 knock-out mice as a model to investigate the structural cortical defects that could lead to clinical feature in patients carrying KIF7 mutation

The KIF7 gene is located on chromosome 15 in human and encodes a 1343 aa protein containing in the N-terminal part, a kinesin motor domain and a GLI-binding domain followed by a Coiled-coil region and in the C-terminal part, a Cargo domain able to bind a diverse set of cargos ⁴⁷. Table 1 summarizes the clinical features associated with mutations targeting either the kinesin or GLI binding domain in the N-terminal part of the protein, the Coiled-coil domain, or the N-terminal Cargo domain. Interestingly, various mutations are associated with the same clinical picture, suggesting that KIF7 mutations, whatever their nature, could lead to protein loss of function, for example by altering the protein structure and solubility as proposed by Klejnot and Kozielski (2012). All patients carrying mutation in the KIF7 gene have developpemental delay (DD) and intellectual deficit (ID) associated with classical defects of ciliopathies such as ventricle enlargement, macrocephaly, corpus callosum (CC) agenesis and molar tooth sign (MTS). Some patients have anatomical cerebral cortex defects as poor frontal development, atrophy, pachygyria and heterotopia ^9,17,18,49 that could participate not only to DD and ID but moreover to seizure as observed in a quarter of patients. In this context, we used a murine model in which the Kif7 gene had been deleted (Kif7 -/-) ³ to investigate the consequence of KIF7 loss of function on the cortex development. Kif7 -/- mice have been previoulsy characterized as dying at birth with severe malformations, including exencephaly in a third of the mutants, skeletal abnormalities (digits and ribs), neural tube patterning defects, microphtalmia, lack of olfactory bulbs and CC agenesis ^3,4,15. In the present study, the KIF7 loss of function was analyzed in a mouse strain with a mixed genetic background in which exencephaly at E14.5 was observed at the same frequency in Kif7 -/- and control embryos. As previously reported, E14.5 Kif7 -/- embryos displayed microphtalmia (Fig. 1A, black arrow) and polydactily (not illustrated). Interestingly, skin laxity previously reported in ciliopathic patients ^50,51 was observed in all mutants (Fig. 1A, white arrow). Comparison of brains from Kif7 -/- embryos and control littermates at E14.5 revealed that Kif7 -/- could be distinguished by the absence of olfactory bulbs (Fig. 1B, white arrows), a thinning of the dorsal cortex allowing the lateral ventricles to be seen through (Fig. 1B, black arrow and Fig. 1C1) and a prominent diencephalon (Fig. 1C1).

Figure 1.

Table 1.

Kif7 invalidation alters the development of the cortex at E14.5

Analyses performed on rostro-caudal series of frontal sections (Fig. 1C1) confirmed that the brain of E14.5 Kif7 -/- embryos exhibited enlarged lateral ventricles (Fig. 1C2) and thinned dorsal cortex (Fig. 1C3) resulting however in unchanged total width and height of the telencephalon (Fig. 1C4,5). KIF7 depletion in E10.5-E11.5 mouse embryos was reported to alter the dorso-ventral patterning of the medial forebrain ¹⁵, in line with the decreased expression of the transcriptional repressor GLI3R ^3,4. GLI3 loss of function ^52,53 has been associated with an abnormal dorso-ventral patterning of the telencephalon. We confirmed here the decreased expression of GLI3R in the cortex of Kif7 -/- embryos (Fig. S1). However, the frontier of expression of GSH2 (Fig. 1D1) and TBR2 (Fig. 1D2), two transcription factors expressed in the ventral and dorsal telencephalon respectively, was preserved in the pallium-subpallium boundary (PSB) at E14.5 in Kif7 -/- embryos, suggesting a rather normal dorso-ventral patterning of the telencephalic vesicles in their lateral side.

The thinning of the dorsal cortex of E14.5 Kif7 -/- embryos convinced us to examine the structural organization of the cortex of Kif7 -/- embryos. At E14.5, the mouse cortex can be described as a stack of three specialized domains: i) the upper/superficial cortical layers containing post-mitotic cells generated in the proliferative zones of the cortex, including TBR1(+) post-mitotic neurons located in the cortical plate (CP); ii) the deep proliferative layers, ventricular and subventricular zones (VZ and SVZ, respectively), located along the lateral ventricle; iii) the intermediate zone (IZ) located between the CP and the proliferative layers, which hosts the radially and tangentially migrating neurons and growing axons. In control embryos, TBR1(+) cells formed a rather regular, radially organized CP surrounded by two thin continuous layers of MAP2(+) neurons located in the upper marginal zone (MZ) and the bottom subplate (SP) respectively (Fig. 2A, left). In Kif7 -/- embryos, the TBR1(+) CP cells appeared more clustered and the thickness of the upper MAP2(+) layer was extremly irregular. MAP2(+) cells were no longer observed below the CP in the dorsal cortex (Fig. 2A, arrow on right panel). Moreover, focal heterotopia characterized by an abnormal accumulation of TBR2(+) cells were observed in the dorsal or lateral cortex of Kif7 -/- embryos, either at the ventricular or pial surface (Fig. S2). TBR2 labels the intermediate progenitors (IP) of principal cells, which normally form a dense layer in the SVZ of the cortex (Fig. 2B1, left). Beside sporadic heterotopia, the TBR2(+) layer constantly showed an abnormal positioning in the dorsal cortex of Kif7 -/- embryos. TBR2(+) cells indeed reached the brain surface (Fig. 2B1, arrow) where they mixed up with post-mitotic TBR1(+) cells instead of keeping a minimal distance with TBR1 cells as observed in control brains (Fig. 2B2, arrow) and in the lateral cortex of Kif7 -/- brains (Fig. 2B2). Therefore, intermediate progenitors and post-mitotic neurons no longer segregated in the dorsal cortex of Kif7 -/- embryos and no IZ was observed (Fig. 2B2, white arrow).

Figure 2.

The loss of Kif7 alters the connectivity between the cortex and the thalamus at E14.5

The intermediate zone (IZ) hosts the radially and tangentially migrating neurons and comprises the growing cortical and thalamic projections ⁵⁴. Corticofugal axons develop from early stages: the trailing process of post-mitotic neurons migrating radially to the CP first elongates tangentially in the IZ and then differentiates as an axon ^55,56. We thus examined whether the defaults observed in the CP and the lack of IZ in the dorsal cortex of Kif7 -/- embryos did associate with developmental abnormalities of the corticofugal projection. We labeled corticofugal axons by inserting small crystals of DiI in the dorsal or lateral cortex in E14.5 paraformaldehyde fixed brains. After DiI had diffused along corticofugal axons, we analyzed labeled axons on coronal sections. In both control and Kif7 -/- brains, CP neurons located in the dorsal or lateral cortex extended axons below the CP toward the PSB (Fig. 3A). DiI injections in the dorsal cortex of control and Kif7 -/- brains (Fig. 3A1) labeled much less axons than injections in the lateral cortex (Fig. 3A2,A3) according to the latero-medial gradient of cortical development. Very remarkably, the cortical bundles labeled from similar cortical regions were always much smaller in Kif7 -/- than in wild type brains (Fig. 3A, compare right and left columns). Whereas dorsal injections labeled large bundles crossing the PSB in control brains, only a few axons reached the PSB in Kif7 -/- brains (Fig. 3A1), showing that corticofugal axons growth was severely hampered in the dorsal cortex of Kif7 -/- embryos. Corticofugal axons labeled from lateral injection sites in control brains crossed the PSB, extended within the embryonic striatum and continued medially in the internal capsule (IC) where they met thalamic axons retrogradely labeled from the lateral cortex (Fig. 3A3, left panel). In Kif7 -/- embryos, lateral injections labeled axons that either spread in the striatum (Fig. 3A2, right panel) or extended ventrally to the cerebral peduncle (Fig. 3A3, right panel, white arrow), a trajectory never observed in control brains. Moreover, no thalamic axons were retrogradely labeled from the lateral cortex. To identify the trajectory of thalamic axons in Kif7 -/- embryos, we immunostained coronal sections of E14.5 brains with antibodies against the Netrin G1a (NG1a), an early marker of thalamocortical axons (TCA, Fig. 3B) ⁵⁷. At E14.5, in both control and Kif7 -/- brains, NG1a antibodies labeled a large population of dorsal thalamic neurons (Fig. 3B). In control brains, thalamic axons made a right angle turn to join the IC. Then they extended to the PSB and some axons entered the lateral cortex (Fig. 3B, left panel). In Kif7 -/- embryos, most thalamic axons stopped their course at the exit of the diencephalon (Fig. 3B, white arrow in right panel) while some of them formed a short and thick bundle oriented ventrally to the surface of the basal telencephalon (Fig. 3B, white arrow in right panel). No evident structural defect could be observed at the telo-diencephalic junction in Kif7 -/- brain (Fig. S3A). Given the complexity of the trajectory of NG1a thalamic axons, we immunostained thalamic axons in whole brains and imaged them after transparisation using a light-sheet microscope. Reconstruction and three-dimension analysis of labeled projections confirmed that thalamic axons made a sharp turn to reach the IC and then navigated straight to the PBS in control brains (Fig. 3C, Fig. S3B and Movie S1). In contrast, most thalamic axons stopped their course after leaving the diencephalon in Kif7-/- embryos, and formed two large bundles, one oriented to the IC (Fig. 3C, white star and Movie S2) and another one oriented caudally (Fig. 3C, white arrow, Fig. S3B and Movie S2). This last projection was never observed in control brains (Movie S1). The present analysis thus identified impaired trajectories of corticofugal and thalamo-cortical projections at E14.5 in the basal forebrain of Kif7 -/- animals, suggesting impaired axonal guidance mechanisms in the basal forebrain.

Figure 3.

Kif7 invalidation alters the cortical distribution of cIN at E14.5

Because cIN are born in the basal forebrain and likely depend on contact/functional interactions with pioneer corticofugal projections for the first stages of their migration ^58,59, we also examined the distribution of cIN in the developing cortex of Kif7 -/- animals. cIN are generated outside of the cortex, in the medial and caudal ganglonic eminences (MGE, CGE) and preoptic area (POA) of the ventral forebrain. They enter the lateral cortex at E12.5 - E13.5, depending on mouse strains, and colonize the whole cortex by organizing two main tangential migratory streams, a superficial one in the MZ, and a deep and large one in the lower IZ/SVZ ^60,61. Intermediate cortical progenitors of the SVZ and meninges strongly express CxCl12 mRNA ⁶², coding for a chemokine essential for cIN to recognize their tangential pathways of migration in the developing cortex. CXCL12 promotes the tangential progression of cIN in the developing cortex and prevents the premature occurrence from switch to tangential to radial mode of migration that is required for cIN to colonize the CP ^62–65. We analyzed the expression of CxCl12 mRNA in the cortex of mutant embryos using in situ hybridization experiments at E14.5, a stage when TBR2(+) SVZ cells show defective positioning in the dorsal cortex of Kif7 -/- embryos. In control brains, CxCl12 mRNA showed a latero-medial gradient of expression in the SVZ, which ended at the frontier with the hippocampus. In contrast, CxCl12 mRNA was no longer expressed in the dorsal cortex of Kif7 -/- embryos (Fig. 4A, black arrow), in the region where TBR2(+) cells starts to position at the brain surface (Fig. 4B1). We thereafter analyzed the cortical distribution of Tomato(+) MGE-derived cIN at E14.5 in control Nkx2.1-Cre;Rosa26-TdTomato transgenic embryos and in Kif7 -/-, Nkx2.1-Cre;Rosa26-TdTomato transgenic mutant embryos (Fig. 4B and see methods). The latero-medial extent of both the superficial and deep tangential migratory streams was significantly shortened in Kif7 -/- brains as compared to control brains (Fig. 4B1, white arrows). Very remarkably, the cIN deep tangential stream stopped in the region where the layer of TBR2(+) cells started its abnormal switch to the surface of the cortex (Fig. 4B1, white arrows) and did not colonize the dorsal cortex (Fig. 4B2). In addition, cIN presented an altered radial distribution in the cortex of Kif7-/- embryos (Fig. 4C). The superficial migratory stream was thinner and denser in the MZ of Kif7 -/- embryos as compared to control (Fig. 4C1, left panel in C2) and the distance between the superficial and deep tangential migratory streams was significantly diminished in the lateral cortex of Kif7 -/- mutants (Fig. 4C2). This decrease is in agreement with the reduced corticofugal projections identified in Kif7 -/- mutants, as these axons navigate tangentially in the IZ. The reduced latero-dorsal distribution of cIN and their abnormal distribution in the cortical thickness reflected structural and molecular abnormalities identified in the cortex of Kif7 -/- embryos. To verify if migratory defaults proper to Kif7 -/- cIN could moreover contribute to their abnormal distribution in the cortex of Kif7 -/- embryos, we analyzed the migration of cIN in living cortical slices.

Figure 4.

Kif7 invalidation and SHH pathway activity both affect the migratory behavior of cIN in cortical slices

The forebrain of E14.5 Nkx2.1-Cre;Rosa26-TdTomato transgenic embryos exhibiting or not a Kif7 deletion (control and Kif7 -/-, respectively) were sliced coronally to image the cortical migration of cIN by time-lapse video-microscopy (Fig. 5). In control slices (Fig. 5A1, Supplementary Movie S3), fluorescent cIN migrated tangentially from the PSB to the dorsal cortex in two main pathways, a superficial one in the MZ and a deep one in the lower IZ/SVZ. All along these pathways, some cIN operated a tangential to oblique/radial migration switch to colonize the CP. Since high cell density in the MZ prevented a dynamic monitoring of tangentially migrating cells, cells migrating in the MZ were not analyzed in this study.A large proportion of cells imaged during the recording session (59.3 %), either maintained tangentially oriented trajectories in the deep stream (28.7 %, red trajectories in Fig. 5A1, left column of Fig. 5A2 and Fig. 5A3) or reoriented to the CP along oblique (and less frequently radial) trajectories (30.6 % green trajectories in Fig. 5A1, left column in Fig. 5A2). A majority of cells reached the CP surface and remained there. A smaller proportion of cIN moved from the deep stream to the opposite - ventricular-side of the slice (11.7%, blue trajectories in Fig. 5A1, left column in Fig. 5A2 and Fig. 5A3). In the VZ, deep stream, and CP, a minimal proportion of cIN (3.6 %) moved very short distances or remained immobile (dark colors in the left column of Fig. 5A2). A significant proportion of cIN (15.6 %) migrated radially, traveling all over the cortex thickness and frequently inverting their polarity in the MZ or at the ventricular surface (pink trajectories in Fig. 5A1, left column in Fig. 5A2 and Fig. 5A3). Finally, 8 % (orange) of the recorded cIN moved backward to the ventral brain and 1.7 % of them (grey) did not show any specific directionality or layer specificity and were classified as chaotic (Fig. 5A1, left column in Fig. 5A2 and Fig. 5A3). A large majority of cIN presented a characteristic saltatory behavior, alternating stops and fast moves ⁶⁶. Analyses of resting and moving phases of the largest cIN population recorded in the deep stream and/or moving to the CP are illustrated in Fig. 5B1-3 (left column of histograms). The trajectories followed by cIN in the cortical slices of Kif7 -/- embryos dramatically differed from those in control slices (Fig. 5A2,A3 and Supplementary Movie S4 compared to S1). The number of cIN migrating tangentially in the deep migratory stream and moving to the CP from the deep stream dropped drastically (17.8 % as compared to 59.3 % in control slices), instead, they were immobile (35.7 % as compared to 3.6 % in control slices) especially in the SVZ/lower IZ. Almost the same proportion of cIN moved to the ventricle and to the CP (10.2 % and 13.9 %, respectively, as compared to 11.7 % and 30.6 % in control slices) suggesting that the radial asymmetry of cortical slice was lost for Kif7 -/- cIN. The proportion of cIN migrating radially across cortical layers remained similar as in control slices (17.2 % versus 15.6 % in control) but the proportion of cIN with chaotic trajectories strongly increased (7.8 % versus 1.7 % in control slices, Fig. 5A2, second column, Fig. 5A3). Another major change observed in the Kif7 -/- cortical slices was a significant decrease of the migration speed (excluding immobile cIN) (Fig. 5B1) and an abnormal saltatory behavior, with less frequent but longer resting phases than in control slices (Fig. 5B2,B3). Of note, trajectories were analyzed at E14.5 in the lateral and latero-dorsal cortex where CxCl12 mRNA was observed in both control and Kif7 -/- embryos (Fig. 4A). Because CXCL12 is a potent chemoattractant for migrating cIN, the very large proportion of immobile cIN recorded in the CXCL12-expressing deep stream of Kif7 -/- slices was suggestive of intrinsic migratory defaults of Kif7 -/- cIN. Kif7 ablation has been shown to induce either transcriptional SHH signaling activation by inhibiting the processing of GLI3 in GLI3-R repressor, or transcriptional SHH signaling inhibition by inhibiting the positive impact of KIF7 on the GLI1/2 dependent SHH signaling ³. We thus examined whether the acute application of drugs able to either promote or inhibit the SHH signaling could induce in wild type cIN migratory defaults resembling those of cIN in Kif7 -/- cortical slices. SHH application on control slices minimaly affected the trajectories of cIN (Fig. 5A2, compare left and right columns, Fig. 5A3), except an increased proportion of cIN migrating radially (29 % compared to 15.6 % in control slices) at the expense of cIN migrating from the deep stream to the CP (20.8 % instead of 30.6 % in control slices). In addition, cIN moving radially across the cortical layers no longer stopped in the CP and frequently moved backward to the VZ (Fig. 5A2,A3, Supplementary Movie S5). The frequency of chaotic trajectories (grey, Fig. 5A2) did not increase, and the migration speed of cIN was slightly decreased compared to control slices (Fig. 5B1). Main dynamic alteration of SHH treated cIN was the increased duration of resting phases (Fig. 5B2,B3). On the contrary, application of the SHH pathway inhibitor cyclopamine strongly altered cIN trajectories (Fig. 5A2,A3). For example, the proportion of immobile cIN and of cIN moving short distance reached 33.7 % as compared to 3.6 % in control slices (Fig. 5A2). Most immobile cells in cyclopamine treated slices were located in the deep stream, and only few cIN switched from the deep stream to the CP at the benefit of cIN migrating backward to the ventral brain. Therefore, inhibiting the SHH pathway mainly affected the ability of cIN to move tangentially in the deep stream and to switch to the CP (Fig. 5A2,A3, Supplementary Movie S6),. Globally, cIN trajectories in cyclopamine treated slices recalled the trajectories of cIN in Kif7 -/- cIN (Fig. 5A2, compare columns 2 and 3), suggesting that Kif7 ablation resembled the cyclopamine-mediated SHH pathway inhibition. Nevertheless, whereas the migration speed of Kif7 -/- cIN was significantly decreased in the deep tangential pathway and not in CP, cyclopamine application decreased the migration speed only in the CP (Fig. 5B1). The strong influence of cyclopamine on the cortical migration of cIN led us to determine the distribution of the endogenous activator SHH in the developing cortex.

Figure 5.

Distribution of Shh mRNA and SHH protein in the E14.5 cortex

Shh mRNA is strongly expressed by the mantle zone of the ganglionic eminences (MGE, CGE) and POA during the time cIN differenciate ^24,56 but its level of expression in cIN after they reach the cortex is controversial ^29,31,67. We thus reexamined Shh mRNA expression in forebrain and SHH protein distribution in cortical layers. In situ hybridization (ISH) experiments performed at E13 confirmed the strong expression of Shh mRNA in the ventral forebrain, especially in the SVZ and mantle zone of the MGE (Fig. 6A1,6B1) in agreement with the SHH-dependent Gli1 mRNA expression in a VZ subdomain of the MGE ⁶⁸. In contrast, Shh transcripts were bearly detectable in the cortex. Using RNAscope, whose sensitivity and spatial resolution is higher compared to standard ISH, we re-examined the expression pattern of Shh mRNA at E14.5. In the ventral forebrain, RNAscope Shh transcript detection showed similar expression pattern as ISH (Fig. 6A2,B2). In addition, the expression domains of Shh mRNA and Lhx-6 mRNA, a cIN marker, overlapped in the mantle zone of the MGE (Fig. 6A2). In the cortex, some cells oriented either radially in the VZ/SVZ or tangentially in the tangential migration pathways expressed Shh mRNA at a very low level (one or two dots versus large cluster in the MGE) (Fig. 6A2). In the tangential migration streams where a large number of cIN expressed Lhx-6 mRNA (Fig. 6C1,C2), cIN that co-expressed Lhx-6 and Shh mRNA represented near 5 % of cIN expressing Lhx-6 transcript (Fig. 6C3).

Figure 6.

Antibodies that recognize the N-ter part of SHH [SHH-Nter, e.g. “activated” SHH ⁶⁹] strongly labeled brain regions known to strongly express SHH ^67,70,71, such as zona limitans intrathalamica and the lateral walls of the third ventricle at E12.5 (Fig. S4), and the choroid plexus at E14.5 (Fig. 6D1), validating the SHH-Nter antibodies used here. At E14.5, SHH-Nter antibodies immunostained bright tiny dots aligned radially in the VZ/SVZ, and tangentially in the IZ. In the CP, dots were again radially aligned and some CP cells presented a rather diffuse cytoplasmic staining, which was not observed in the bottom layers (Fig. 6D2). We observed no particular SHH enrichment in the lower IZ/SVZ where cIN migrate, suggesting that their contribution to the cortical pattern of SHH expression was minimal. Moreover, large dots were observed in the subpallium at the ventricular angle (Fig. 6D4). Together, our ISH and immunostaining experiments showed that the cortical SHH protein has probably an extrinsic origin as previously described at the early stage of cerebellum development (Huang et al, 2010).

Ontogeny of Kif7 mutant brain developmental defects

After E14.5, the cortex of Kif7 -/- embryos continued to develop. Analyses performed at E16.5 showed that cIN continued to migrate tangentially and reached the dorsal cortex in Kif7 -/- embryos (Fig. 7A). Nevertheless, the tangential migration of cIN to the dorso-medial cortex remained delayed in Kif7 -/- embryos with regard to the tangential migration of cIN in control embryos. Kif7 -/- cIN failed to reach the hippocampus identified by the presence of TBR2(+) cells, as observed in control brains (compare left and right panels in Fig. 7A). At E16.5, NG1a(+) thalamic axons colonized the whole medial and rostral cortex, in both control and Kif7 -/- embryos (Fig. 7B). Nevertheless, thalamic axons were less dense in Kif7 -/- than in control cortex and exhibited abnormal trajectories (Fig. 7C) toward the cortical plate. Analyses performed in a surviving P0 animal (Fig. S5) revealed an abnormal distribution of cIN in the cortical plate, with a decreased density in the bottom layers, which were moreover thinner than in control brains, as illustrated by CTIP2(+) staining (Fig. S5A). The cortical distribution of thalamic axons was extremely perturbed in Kif7 -/- brains: a dense bundle of NG1a(+) fibers ran below the cortical plate to the dorsal cortex and failed to invade the CP (Fig. S5B).

Figure 7.

Discussion

Our aim in the present paper was to better understand the developmental origin of functional abnormalities (e.g. epilepsy, cognitive deficits, …) in cortical circuits of human patients with KIF7 mutations by characterizing the developmental abnormalities of the cerebral cortex in a Kif7 -/- mouse model. We show that Kif7 ablation affects the development of the two populations of cortical neurons, cortical plate neurons and GABAergic interneurons, in specific and distinct ways, accordingly with their dorsal and ventral origin in the telencephalon. The alterations of the CP development (lack of most subplate cells in the dorsal cortex, and delayed corticofugal projection) are remiscient of defaults previously described in GLI3 null mutants that lack most subplate neurons and pioneer cortical projections ⁷². And the abnormal distribution of Kif7 -/- cIN in the developing cortex indeed reflects the structural and functional alterations of cortical layers on which cIN migrate (e.g. abnormal CXCL12 expression in the SVZ). However, we identify moreover migration defaults of Kif7 -/- cIN that recall those of wild type cIN treated with the SHH pathway inhibitor cyclopamine, suggesting that the SHH pathway activity is inhibited in Kif7 -/- cIN. Kif7 ablation thus alters specifically and differently the SHH pathway activity in CP and cIN subpopulations, in agreement with their dorsal or ventral origin.

Kif7 knock-out mice as a model to study the effect of mutation in the KIF7 gene in human

The deletion of Kif7 gene in mice leads to lethality the first day of life, but up to birth, the brain develops and a previous study demonstrated that Kif7 mutant mice was a good model to dissect the mechanism that led to corpus callosum agenesis in patients ¹⁵. All patients carrying mutation in the KIF7 gene on both alleles have developmental delay/intellectual disability (ID) and some have seizures. Investigations on cerebral malformation by MRI have shown that some of them have alterations in the cerebral cortex including cortical atrophy ^9,10,18 and pachygyria ¹⁷. Detecting defects in the cerebral cortex is more demanding in MRI quality images that corpus callosum agenesis or molar tooth sign, we can speculate that the number of patients with cerebral cortex defects was underscored. Malformations of cortical development was recognized as causes of ID ⁷³ and epilepsy ⁷⁴ and the cellular defects underlying these pathologies could imply defects in cell proliferation, neuronal migration and differenciation that lead to improper excitatory/inhibitory balance, connectivity into the cortex or of the cortex with other brain structures. The mechanism of action of KIF7 to transduce SHH ciliary dependent signaling has been debated. It has been accepted that SHH activates KIF7 binding to microtubules and its accumulation to the tip of the cilium, making the kinesin motor domain the most essential domain for SHH transduction. A more recent study proposed that KIF7 is an immobile kinesin translocated to the cilium tip after SHH binding by a complex KIF3A/Kif3B/KAP ⁷⁵ suggesting that domains in the KIF7 gene essential to its function are more complex. This could explain why any mutation, even point mutation in the Coiled-coil domain or in C-terminal part of the KIF7 protein induces clinical feature as severe as when mutation led to a highly truncated protein. Another explanation is that any mutation causes unproper folding of the protein inducing its degradation by the proteasome.

KIF7 loss of function alters the cerebral cortex organization and the connectivity between the cortex and thalamus

In the mouse embryo, GLI3 plays a major role in patterning the dorsal telencephalon and in regulating the proliferation and differenciation of cortical neurons ^40–42. The Kif7 knock-down that likely decreases GLI3R level leads to a decrease of apical radial progenitors and basal IP divisions, and of neuronal differenciation ⁴⁴. In fibroblasts isolated from lung of patients carrying KIF7 mutation and in E11.5 Kif7 -/- whole embryo, the processing of GLI3-full length in GLI3-R is decreased and GLI-A increased leading to over-SHH signaling ^3,49. Our study demonstrates that the level of GLI3-R was decreased in the developing cortex of the Kif7 -/- embryos, whose layering of principal neurons was altered and exhibited sporadic heterotopia and delayed connectivity between the cortex and the thalamus. Heterotopia have been observed in one patient ¹⁷ and we found cortical heterotopia in some Kif7 -/- brains. Cortical heterotopia is not a landmark of ciliopathy but were detected in a Joubert syndrom patient carrying a mutation in the CELSR2 gene coding for a planar cell polarity protein ⁷⁶ essential for neural progenitor cell fate decision, neural migration, axon guidance and neural maturation ^77,78. In the patient as in the mice model, the heterotopia are focal and may have been missed in other patients. They nevertheless emphasize the mistrafficking of principal cells in KIF7 depleted brain. Although disorganized thalamo-cortical connectivity had never been reported in patients with KIF7 mutation or in patients with ciliopathy, thalamocortical connectivity is essential for cognitive performances in young infants ⁷⁹ and its alteration leads to neurodevelopemental delay and later cognitive deficits ⁸⁰. Moreover, a link beween ciliary function, cortex development and connectivity with the thalamus had been previously identified in mutant mice lacking planar cell polarity proteins CELSR2 and FZD3. These mice display ciliary defects and an abnormal development of tracts between the thalamus and the cortex leading to an hypotrophy of the intermediate zone where thalamo-cortical and corticofugal axons navigate^81,82.

Extra-cortical origin of SHH

Beside its major function in forebrain patterning ^20,40, SHH moreover regulates the embryonic and perinatal cerebral cortex development ^27,28. The origin of the morphogen in this brain structure remains obscure. In agreement with a recent set of data from Moreau et al. ⁸³, we showed that cIN born in the MGE or POA no longer express SHH after they reach the developing cortex. In addition, the level of local Shh mRNA synthesis was extremely low in the cortex at E14.5. In contrast, SHH protein was detected in immunoposive dots distributed throughout the cortex. Dots aligned radially in the VZ and CP, and tangentially in the IZ. This cortical distribution is compatible with an extrinsic origin of SHH and transventricular delivery as described in the cerebellar ventricular zone ⁸⁴. SHH is indeed present in the cerebrospinal fluid and likely secreted by the plexus choroid, since Shh mRNA is expressed in the choroid plexus of mice from E12 to E14.5 ⁸⁵. Accordingly, we showed here that the choroid plexus of the lateral ventricles was strongly immunopositive for SHH at E14.5. Interestingly, the CDO/BOC co-receptor - that favors SHH transport - was also detected along radially oriented structures by immunostaining in the mouse embryonic cortex (not illustrated). SHH could thus be transported away from the ventricular zone and/or meninges in vesicular structures able to diffuse accross the cortical thickness. Accordingly, it has been shown that SHH transport needs ESCRT-III member protein CHMP1A ¹³ and SHH related proteins are found in exosomes secreted from Caenorhabditis elegans epiderman cells ⁸⁶, Drosophila wing imaginal discs ⁸², chick notochord cells and human cell lines ⁸⁷. How SHH is transported thoughout the cortex should be further explored. It has already be shown that SHH is present in multivesicular bodies, in neurites and filipodia in the post natal hippocampus and cerebellum, and in vesicles located inside and outside of hippocampal neurons in culture ⁸⁸.

Kif7 deletion leads to defects in cortical interneuron migration

The defects induced by gene mutations responsible of primary cilium dysfunction, including Kif7 deletion, have been examined in principal cells born in the dorsal telencephalon, which organize the cortical layers ^89,90. However, the cIN are born ventrally and migrate to the cortex during the embryonic development. Their primary cilium is functional and can transduce local SHH signals to influence their directionality, especially the tangential to radial reorientation of their trajectories toward the cortical plate ²⁹. In the present study, we showed that acute SHH application on control cIN slightly increased the percentage of radially migrating cIN. In contrast, cyclopamine that inhibited the SHH pathway blocked the migration of a large population of cIN in the deep cortical layersdemonstrating that endogeneous cortical SHH is likely to activate the SHH pathway in migrating cIN to control their trajectories. Since Kif7 -/- cIN showed a migratory behavior that recalled the migratory behavior of cyclopamine treated control cIN, we concluded that SHH signaling was inhibited in Kif7 -/- cIN. By consequence, we propose that Kif7 -/- cIN, which differentiate in a ventral telencephalic domain expressing high SHH level, present reduced Gli-A signaling, e.g. decreased SHH signaling. In addition to this cell autonomous defect that impairs cIN migration, the colonization of the dorsal cortex by Kif7 -/- cIN could be delayed due to the decreased dorsal expression of CXCL12 in Kif7 -/- embryos ^62,91.

In conclusion, using the Kif7 -/- murine model, we show that the Kif7 deletion is responsible for a large range of developmental defects in the cerebral cortex, which include: i) alteration of the inhibitory interneurons migration by a combination of intrinsic and extrinsic factors, ii) abnormal formation of cortical layers by the cortical plate neurons and iii) major pathfinding defects in axonal projections between the cortex and the thalamus. Patients carrying mutations in the KIF7 gene are classified as ciliopathic patients and display developmental delay, ID and epilepsy. These clinical features could thus be caused by an abnormal distribution of excitatory and inhibitory cortical neurons leading to impaired excitatory/inhibitory balance, and by the development of abnormal connections in the cerebral cortex and with others brains structures. By demonstrating focal heterotopia and abnormal thalamo-cortical connectivity in the murine model, we hope to open a new field of clinical investigations using MRI and tractography to identify such defects in patients carrying mutations in the KIF7 gene.

Material and methods

Mice

E14.5 embryos of the swiss (for in situ hybridization and RNAscope) and C57/Bl6J (for sonic hedgehog immunostaining) strains were obtained from pregnant females purchased at JanvierLabs (France). Male and female wild type (WT) and Kif7 -/- animals (E14.5, E16.5 and P0) expressing TdTomato or not in the MGE-derived cIN (Nkx2.1-Cre;Rosa26-TdTomato) were generated in our animal facility by mating male Kif7 +/-; Nkx2.1-Cre;Rosa26-TdTomato and female Kif7 +/- mice to produce embryos and P0. Thirty four pregnant females were used. Kif7 +/- mice are a generous gift of Pr Chi-Chung Hui (the Provider, SickKids Hospital, Toronto, USA) and Prof Bénédicte Durand (the Transferor, CNRS, Lyon, France). Mice were genotyped as described previously ³. The day of the vaginal plug was noted E0.5. Experiments have been validated and approved by the Ethical committee Charles Darwin (C2EA-05, authorized project 02241.02) and mice were housed and mated according to European guidelines. Both male and female animals were used in this study. Sex is not considered as a biological variable in embryos.

Immunohistochemistry and imaging

Heads of embryos and P0 animals were fixed by immersion in 0.1% picric acid /4% paraformaldehyde (PAF) in phosphate buffer (PB) for 4 hours and then in 4% PAF in PB overnight. After PBS washes, brains were dissected, included in 3% type VII agarose (Sigma, A0701) and sectionned at 70 μm with a vibratome. Immunostaining was performed on free-floating sections as explain previously (Baudoin et al, 2012). Primary antibodies were goat anti SHH-Nter (1:100, R&D system AF464), goat anti Netrin G1a (NG1a) (1:100, R&D system AF1166), rabbit anti TBR1 (1:1000, Abcam ab31940), rabbit anti TBR2 (1:1000, Abcam ab23345), rabbit anti PAX6 (1:100, clone poly19013, Covance PRB-278P), rabbit anti GSH2 (1:2000, Millipore ABN162), chicken anti MAP2 (1:500, Novus, NB30213), and rat CTIP2 (1:1000, Abcam ab18465). Primary antibodies were incubated in PGT (PBS/gelatine 2g per L/TritonX-100 0.25%) with 0.1% glycine and 0.1% lysine for anti Netrin G1a antibodies and 3% BSA for anti SHH-Nter antibodies. Primary antibodies were revealed by immunofluorescence with the appropriate Alexa dye (Molecular Probes) or Cy3 (Jackson laboratories) conjugated secondary antibodies diluted in PGT (1:400). Bisbenzimide (1/5000 in PBT, Sigma) was used for nuclear counterstaining. Sections were mounted in mowiol/DABCO (25mg/mL) and imaged on a macroscope (MVX10 olympus) or an epifluorescence microscope (LEICA DM6000) using X40 and X63 immersion objectives, or a confocal microscope (Leica TCS SP5) using a x40 objective. Double immunotraning were performed when possible and images were representative of what was observed in more than 4 animals per genotype except for heterotopia. Macroscop images were treated to remove background around sections using wand tool in Adobe Photoshop 7.0 software. On epifluorescence and confocal microscop images, acquisition parameters and levels of intensity in Adobe Photoshop 7.0 software were similar for WT and Kif7 -/-. Merged images were acquired on the same section except for TBR1 with TBR2 aquired on adjacent sections. The fluorescence intensity along the depth of the cortex was assessed using the plot profile function of ImageJ under a 500 pixels wide line starting in the ventricle to the surface of the brain.

In situ hybridization

Specific antisense RNA probe for Shh, Gli1 and CxCl12 genes (gift of Marie-Catherine Tiveron) were used for in situ hybridization analyses. DIG-probes were synthesized with a labeling kit according to the manufacturer’s instructions (Roche, France). In situ hybridization was performed according to a modification of Tiveron et al ⁴⁵ (see Supplementary material). Sections were mounted on glass slides, dried, dehydrated in graded ethanol solutions, cleared in xylene and coverslipped with Eukitt.

RNAscope

Embryonic E14.5 heads were cuted in cold Leibovitz medium (Invitrogen) and immersion fixed in cold 4% (w/v) paraformaldehyde (PFA) in 0.12 m phosphate buffer, pH 7.4, overnight. Brains were then cryoprotected in PFA 4% /sucrose 10 %, embedded in gelatin 7.5% /sucrose 10% at 4°C and frozen. Biological samples were kept at -80°C until coronally sectioned at 20 μm with cryostat. RNAscope experiment was performed according manufacturer’s instructions (RNAScope^® Multiplex Fluorescent V2 Assay, ACDbio) after a pre-treatment to remove gelatin. Two different probes/channels were used (C1 for Shh, C2 for Lhx-6). Probes were diluted according manufacturer’s instructions (1 Vol of C2 for 50 vol of C1) and dilution of the fluorophores (Opal 520 and 570) was 1:1500. Sections were mounted in mowiol/DABCO (25mg/mL) and imaged on a confocal microscop (Leica TCS SP5) using an immersion X10 or X63 objectives on 10 µm stacks of images (1 image /µm).

DiI experiments

Tiny crystals of DiI (1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate) were inserted in the dorsal or lateral cortex of WT and Kif7 -/-embryonic brains fixed at E14.5 by immersion in 4% PAF. Injected brains were stored in 4% PAF at room temperature in the dark for several weeks to allow DiI diffusion. Before sectioning, brains were included in 3% agar. Coronal sections 60 μm thick were prepared with a vibratome and collected individually in 4% PAF. Sections were imaged with a macroscope (MVX10 Olympus).

Whole brain clearing and imaging

E14 and E16 embryos (WT, n=5; Kif7 -/-, n=6) were collected in cold L15 medium, perfused transcardially with 4% PAF using a binocular microscope. Brains were dissected and postfixed 3 days in 4% PAF at 4°C, and stored in PB at 4°C until clearing. All buffer solutions were supplemented with 0.01% Sodium Azide (Sigma-Aldrich). Whole brain immunostaining and clearing was performed at RT under gentle shaking according to a modified version of the original protocol ⁴⁶. Briefly, perfused brains were dehydrated in graded methanol solutions (Fischer Scientific) (20, 50, 80% in ddH₂O, 2×100%, 1h30 each), bleached overnight in 3% hydrogen peroxide (Sigma-Aldrich) in 100% methanol and rehydrated progressively. After PBS washes, brains were blocked and permeabilized 2 days in PBGT (0.2% gelatin (Merck), 0.5% triton X-100 (Sigma-Aldrich) in PBS). Brains were incubated 3 days at 37°C in primary goat anti Netrin G1a (1:100, R&D system AF1166) and rabbit anti Tbr1 (1:1000, Abcam ab31940) antibodies. After 1 day of PBGT washes, brains were incubated 1 day at 37°C in secondary 647 donkey anti-goat (1:1000, Molecular probes,) and cy3 donkey anti-rabbit (1:1000,Jackson laboratories) antibodies.Immunolabeled brains were washed 1 day in PBS, embedded into 1.5% low-melting agarose (type VII, Sigma) in 1% ultra-pure Tris-acetate-EDTA solution, placed in 5-ml tubes (Eppendorf, 0030119452) and dehydrated 1h in each methanol baths (50%, 80% and 2×100%). Samples were then incubated for 3h in 33% methanol / 66% dichloromethane (DCM, Sigma-Aldrich), washed in 100% DCM (2×30 min) and incubated overnight (without shaking) in dibenzyl ether (DBE). Brains were stored in DBE at RT. Cleared samples were imaged on a light-sheet microscope (LaVision Biotec, x6.3 zoom magnification) equipped with a sCMOS camera (Andor Neo) and Imspector Microscope controller software. Imaris (Bitplane) was used for 3D reconstructions, snapshots and movies.

Brain slices

We dissected brains of E14.5 WT and Kif7 -/- transgenic Nkx2.1-cre, TdTomato embryos in cold L15 medium, embedded in 3% type VII agarose and sectioned coronally with a manual slicer as explained in Baudoin et al, 2012. Forebrain slices 250 µm thick were transferred in Millicell chambers and cultured for 4 hours in F12/DMEM medium supplemented with CFS 10% in a CO2 incubator (Merck Millipore) prior to pharmacological treatment and recording. Slices were incubated in drug for 2 hours before transfer in culture boxes equipped with a bottom glass coverslip for time-lapse imaging.

Pharmacological treatment

Either recombinant Mouse SHH (C25II), N-terminus (464-SH-025, R&D systems) at 0.5µg/mL final or cyclopamine (C4116, Sigma) at 2.5 µM final were added to the culture medium of slices and renewed after 12 hours. Recombinant SHH was reconstituted at 100 µg/mL in sterile PBS with BSA 0.1%. The stock solution of cyclopamine was 10 mM in DMSO and the culture medium of control experiments contained 1/4000 DMSO (vehicle). Control experiments in culture medium with and without vehicle did not differ and were analyzed together.

Videomicroscopy and image processing

Slices were imaged on an inverted microscope (Leica DMI4000) equipped with a spinning disk (Roper Scientific, USA) and a temperature-controlled chamber. Multi-position acquisition was performed with a Coolsnap HQ camera (Roper Scientific, USA) to allow the recording of the whole cortex. Images were acquired with a X20 objective (LX20, Fluotar, Leica, Germany) and a 561 nm laser (MAG Biosystems, Arizona). Z-stacks of 30 μm were acquired 50 µm away from the slice surface, with a step size of 2 μm and a time interval of 2 or 5 minutes for at least 21 hours. Acquisitions were controlled using the Metamorph software (Roper Scientific, USA). Cell trajectories were reconstructed on movies by tracking manually the cell rear with MTrakJ (ImageJ plugin, NIH, USA) and were clustered according to their position in cortical layers and to their orientation (see details in Fig. 5). The migration speed of cells and the frequency and duration of pauses were extracted from tracking data using an excel macro.

Study design

WT group was compared to Kif7 -/- group and to pharmacological treatment groups for brain slice experiments. The experimental unit was a single animal for immunohistochemistry analysis and individual cell for videomicroscopy analysis.

Statistical analysis

All data were obtained from at least three independent experiments and are presented as mean ± SEM (standard error of mean). Statistical analyses were performed with the GraphPad Prism software or R. Statistical significance of the data was evaluated using the unpaired two-tailed t test, the Mann–Whitney test, the Chi2 test or the Two-way ANOVA test followed by a post-hoc test. Data distribution was tested for normality using the D’Agostino and Pearson omnibus normality test. Values of P<0.05 were considered significant. In figures, levels of significance were expressed by * for P<0.05, ** for P<0.01, *** for P<0.001 and **** for P<0.0001.

Data availabitily

The data presented in this study are available from the corresponding author upon request without undue reservation.

Acknowledgements

Marie-Christine Tiveron is acknowledged for the gift of the CXCL12 expression vector. We thank all members of the Métin’s Team for constructive discussions. We gratefully acknowledge the Imaging plateform facility of the Fer à Moulin Institute for the use of their microscopes, and the animal facility of the Fer à Moulin Institute for animal care and breeding.

Funding

This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Sorbonne University. Agence Nationale de la Recherche (ANR, grant MIGRACIL to C.M.), Fondation pour la Recherche sur le Cerveau (grant R11080DD to C.M.), Fondation J. Lejeune (grant R14108DD to C.M.).

Author contributions

CM and JM conceptualized the project and supervised the work. MP, VG, SSL, JP, AM, NR, CM and JM perfomed experiments. MP, SSL, AM, NR, CM and JM analyzed the data. MP, VG, SSL, CM and JM wrote the paper.

We declare no Competing interest.

Supplementary methods

Brain lysate preparation and immunoblotting

Brains from WT and Kif7 -/- were dissected at E14.5 and cortex and MGE were separately sonicated in boiling 1% SDS. Equal amounts of proteins (40 μg) were loaded on NuPAGE 4–12% Bis-Tris gels (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to 0.45 μm Nitrocellulose membranes. Membranes were cut to detect signals from proteins above and below 55KDa and were blocked with 50 g/l non-fat dry milk in PBS/0.1% Tween 20 for 1h at RT, incubated with primary antibodies in the same solution O/N at 4°C, 1 h at RT with appropriate IRDye-conjugated secondary antibodies, and imaged and quantified using Odyssey Imaging System (LiCOR Biosciences, Lincolm, NE, USA). The commercial antibodies were from the following sources: anti-GLI3 (R&D Sytem-Biotechne, goat, #AF369, 1:200), anti-GAPDH (In vitrogen, mice, #AM4300, 1:2.000), Donkey-anti-goat-IR800 (Advansta, R-05781, 1:20.000), Goat-anti-mouse-IR700 (Advansta, R-05055, 1:20.000).

In situ hybridization

Fixed embryos were cryoprotected overnight in PBS with 10% sucrose, embedded in OCT (Tissue-Tek, Miles, Elkhart, IN) and frozen on dry ice. 10 µm cryostat sections were thaw-mounted on Superfrost slides (Menzel-Gläser), left to dry at room temperature (RT), and stored at -80°C. Thawed sections were treated two X 10 min in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, 1 mM EDTA, 50 mM Tris, pH 8.0), postfixed in 4% PFA for 10 min at RT, and washed three X 5 min with PBS. The slides were then transferred in 100 mM triethanolamine, pH 8.0 for 2 min, and then acetylated for 10 min at RT by adding dropwise acetic anhydride (0.25% final concentration) while being rocked, and washed again three X 5 min in PBS. The slides were prehybridized briefly with 500 µl of hybridization solution (50% formamide, 5x SSC, 5x Denhardt’s, 500 mg/ml herring sperm DNA, 250 mg/ml yeast RNA) and hybridized overnight at 70°C with the same solution in the presence of the heat-denatured DIG-labeled RNA probe. The following day, slides were washed in posthybridization solution (50% formamide, 2x SSC, 0.1% tween20) at 70°C first until coverslips slid off, then twice for 60 min at 70°C and finally at RT for 5 min. Slides were washed with buffer 1 (100 mM maleic acid, pH 7.5, 150 mM NaCl, 0.05% Tween 20), blocked for 30 min in buffer 2 (10% heat-inactivated horse serum in buffer 1), incubate overnight at 4°C with alkaline phosphatase-coupled anti-DIG antibody (Roche Diagnostics, Mannheim, Germany) diluted 1:1000 in buffer 2, rinsed twice for 5 min with buffer 1, and equilibrated for 30 min in buffer 3 (100 mM Tris, pH 9.5, 100 mM NaCl, 50 mM MgCl2). The signal was visualized by a color reaction using 250 µl of buffer 4 per slice [6.6 µl/ml NBT (4-nitroblue tetrazolium chloride, Roche Diagnostics), 3.3 µl/ml BCIP (5-bromo-4-chloro-3-indoyl-phosphate, Roche Diagnostics) in buffer 3]. The color reaction was allowed to develop in the dark at RT during few hours and was stopped with PBS.

Supplementary figures

Figure S1.

Figure S2.

Figure S3.

Figure S4.

Figure S5.