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A transient role of the ciliary gene Inpp5e in controlling direct versus indirect neurogenesis in cortical development

  1. Kerstin Hasenpusch-Theil
  2. Christine Laclef
  3. Matt Colligan
  4. Eamon Fitzgerald
  5. Katherine Howe
  6. Emily Carroll
  7. Shaun R Abrams
  8. Jeremy F Reiter
  9. Sylvie Schneider-Maunoury
  10. Thomas Theil  Is a corresponding author
  1. Centre for Discovery Brain Sciences, University of Edinburgh, United Kingdom
  2. Simons Initiative for the Developing Brain, University of Edinburgh, United Kingdom
  3. Sorbonne Université, CNRS UMR7622, INSERM U1156, Institut de Biologie Paris Seine (IBPS) - Developmental Biology Unit, France
  4. Department of Biochemistry and Biophysics, University of California, San Francisco, United States
  5. Chan Zuckerberg Biohub, United States
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Cite this article as: eLife 2020;9:e58162 doi: 10.7554/eLife.58162

Abstract

During the development of the cerebral cortex, neurons are generated directly from radial glial cells or indirectly via basal progenitors. The balance between these division modes determines the number and types of neurons formed in the cortex thereby affecting cortical functioning. Here, we investigate the role of primary cilia in controlling the decision between forming neurons directly or indirectly. We show that a mutation in the ciliary gene Inpp5e leads to a transient increase in direct neurogenesis and subsequently to an overproduction of layer V neurons in newborn mice. Loss of Inpp5e also affects ciliary structure coinciding with reduced Gli3 repressor levels. Genetically restoring Gli3 repressor rescues the decreased indirect neurogenesis in Inpp5e mutants. Overall, our analyses reveal how primary cilia determine neuronal subtype composition of the cortex by controlling direct versus indirect neurogenesis. These findings have implications for understanding cortical malformations in ciliopathies with INPP5E mutations.

Introduction

Building a functional cerebral cortex which confers humans with their unique cognitive capabilities requires controlling the proliferation of neural progenitor cells and the timing and modes of neurogenic cell divisions. Varying the timing and modes of neurogenesis affects neuronal numbers and subtype composition of the cortex (Florio and Huttner, 2014). In the developing murine cortex, radial glial cells (RGCs) represent the major neural stem cell type. Residing in the ventricular zone, they express Pax6 and undergo interkinetic nuclear migration dividing at the ventricular surface (Götz et al., 1998; Warren et al., 1999). Initially, RGCs go through rounds of symmetric proliferative divisions to produce two RGCs increasing the progenitor pool but switch to asymmetric divisions at the beginning of cortical neurogenesis. RGCs generate neurons in two ways, either directly or indirectly via the production of basal progenitors (BPs) that settle in the subventricular zone (SVZ) and express the Tbr2 transcription factor (Englund et al., 2005). In the mouse, the majority of BPs divide once to produce two neurons, whereas the remainders undergo one additional round of symmetric proliferative division before differentiating into two neurons (Haubensak et al., 2004; Miyata, 2004; Noctor et al., 2004). In this way, BPs increase neuron output per RGC and have therefore been implicated in the evolutionary expansion of the mammalian cerebral cortex (Martínez-Cerdeño et al., 2006). Thus, the balance between direct and indirect neurogenesis is an important factor in generating appropriate neuron numbers and types.

The mechanisms that fine tune this balance and thereby adjust the numbers and types of neurons produced in the cortex have only recently been investigated. Mitotic spindle orientation (Postiglione et al., 2011) and endoplasmic reticulum (ER) stress (Gladwyn-Ng et al., 2018; Laguesse et al., 2015) are contributing factors to control the generation of basal progenitors. In addition, levels of Slit/Robo and Notch/Delta signaling were shown to be evolutionarily conserved factors that determine the predominant mode of neurogenesis (Cárdenas et al., 2018). Moreover, feedback signals from postmitotic neurons control the fate of radial glial daughter cells involving the release of Neurotrophin-3 and Fgf9 (Parthasarathy et al., 2014; Seuntjens et al., 2009) as well as the activation of a Notch-dependent signaling pathway (Wang et al., 2016). These studies highlight the importance of cell-cell signaling in controlling the cell lineage of cortical progenitors (Silva et al., 2019) and emphasize the necessity of studying the cellular mechanisms by which these signals control the decision by RGCs to undergo direct or indirect neurogenesis.

Given the importance of cell-cell signaling, it is likely that the primary cilium, a signaling hub in embryogenesis in general and in neural development in particular (Valente et al., 2014), plays key roles in determining the balance between direct versus indirect neurogenesis. The cilium is a subcellular protrusion that predominately emanates from the apical surface of RGCs projecting into the ventricular lumen. The phenotypes of several mouse lines mutant for ciliary genes underline the importance of the primary cilium in forebrain development but these mutants often suffer from severe patterning defects (Ashique et al., 2009; Besse et al., 2011; Willaredt et al., 2008) which make elucidating ciliary roles in determining the lineage of cortical progenitors difficult. To address how cilia control cortical progenitor development, we investigated corticogenesis in a mouse mutant for the ciliary gene Inpp5e.

INPP5E is mutated in Joubert syndrome (JS) (Bielas et al., 2009; Jacoby et al., 2009), a ciliopathy characterized by cerebellar defects in which a subset of patients also shows malformations of the cerebral cortex including heterotopias, polymicrogyria and agenesis of the corpus callosum (Valente et al., 2014). Inpp5e encodes Inositol polyphosphate 5 phosphatase E, an enzyme that is localized in the ciliary membrane and that hydrolyses the phosphatidylinositol polyphosphates PI(4,5)P2 and PI(3,4,5)P3 (Bielas et al., 2009; Jacoby et al., 2009). In this way, it controls the inositol phosphate composition of the ciliary membrane and thereby regulates the activity of several signaling pathways and cilia stability (Bielas et al., 2009; Chávez et al., 2015; Garcia-Gonzalo et al., 2015; Jacoby et al., 2009; Plotnikova et al., 2015). In contrast to Inpp5e’s extensively studied biochemical and cellular roles, little is known how these diverse functions are employed at the tissue level to control RGC lineage.

Here, we show that loss of Inpp5e function results in an increase in neuron formation at the expense of basal progenitor production in the E12.5 cortex and in an overproduction of Ctip2+ layer V neurons in newborn mutants. Moreover, RGC cilia show unusual membranous structures and/or abnormal numbers of microtubule doublets affecting the signaling capabilities of the cilium. The levels of Gli3 repressor (Gli3R), a critical regulator of cortical stem cell development (Hasenpusch-Theil et al., 2018; Wang et al., 2011), is reduced and re-introducing Gli3R rescues the decreased formation of basal progenitors. Taken together, these findings implicate Inpp5e and the primary cilium in controlling the decision of RGCs to either undergo direct neurogenesis or to form basal progenitors, thereby governing the neuronal subtype composition of the cerebral cortex.

Results

Inpp5eΔ/Δ embryos show mild telencephalic patterning defects

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 Inpp5eΔ/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 Inpp5eΔ/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.

Inpp5e controls direct vs indirect neurogenesis in the lateral neocortex

Based upon these findings, we started analyzing the proliferation and differentiation of RGCs in Inpp5eΔ/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).

Figure 1 with 3 supplements see all
Increased neuron formation in the dorsolateral telencephalon of E12.5 Inpp5Δ/Δ embryos.

(A–D) Pax6/PCNA double immunofluorescence staining revealed the proportion of apical radial glial cells which remained unaltered in the mutant. The boxes in (A) indicate the regions in the medial (m) and lateral (l) telencephalon at which cell counts were performed. (E–H) Reduced proportions of basal progenitors in the lateral telencephalon as revealed by staining for Tbr2 and PCNA. (I–L) Tbr1 immunostaining showed that the proportion of neurons is increased in the lateral telencephalon. (A–J) The insets labelled with ’ and ” are representative magnifications of medial and lateral levels, respectively. All statistical data are presented as means ± 95% confidence intervals (CI); unpaired t-tests; n = 4 except for (H) with n = 5; *p<0.05; **p<0.01. Scale bar: 100 μm (A) and 50 μm (A’). ctx: cortex; LGE: lateral ganglionic eminence.

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 Inpp5eΔ/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 Inpp5eΔ/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.

Proportions of radial glial cells, basal progenitors and neurons in the neocortex of E14.5 Inpp5eΔ/Δ embryos.

(A–D) The proportion of radial glial cells remains unaffected by the Inpp5e mutation as revealed by Pax6/PCNA double immunofluorescence. The boxes in A indicate the regions in the medial (m) and lateral (l) telencephalon at which cell counts were performed. (E–H) Tbr2/PCNA double staining showed a reduced proportion of basal progenitors in the Inpp5eΔ/Δ medial but not lateral neocortex. (I–N) The proportion of Tbr1+Ctip2+ neurons is not significantly altered (I–L), whereas the proportion of Tbr1-Ctip2+ neurons is decreased and increased in the medial and lateral neocortex, respectively. Arrows in (I and J) label Tbr1-Ctip2+ neurons and arrowheads Tbr1+Ctip2+ neurons. (A–J) The insets labeled with ’ and ” are representative magnifications of medial and lateral levels, respectively. All statistical data are presented as means ± 95% confidence intervals (CI); Unpaired t-tests (C, D, H, K–N) and Mann Whitney test (G); n = 4; *p<0.05; **p<0.01. Scale bars: 100 μm (A) and 50 μm (A’, E’ and I’). ctx: cortex; LGE: lateral ganglionic eminence.

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 Inpp5eΔ/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 Inpp5eΔ/ΔRGCs.

Figure 3 with 3 supplements see all
Increased neurogenesis at the expense of basal progenitor formation in the E12.5 Inpp5eΔ/Δ mutant lateral telencephalon.

Immunohistochemistry on sections of E12.5 control (A, D) and Inpp5eΔ/Δ embryos (B, E) that were treated with BrdU 24 hr earlier. (A–C) Tbr2/BrdU double labeling showed that less basal progenitors formed from the BrdU-labeled progenitor cohort in Inpp5eΔ/Δ embryos. (D–F) The proportion of newly formed Tbr1+ neurons was increased in Inpp5eΔ/Δ embryos. The arrows in D and E label Tbr1+BrdU+ cells. All statistical data are presented as means ± 95% confidence intervals (CI); unpaired t tests; n = 4; *p<0.05; **p<0.01. Scale bar: 50 μm.

Cortical malformations in Inpp5eΔ/Δ embryos

Next, we investigated the consequences of this increase in direct neurogenesis on cortical size and layer formation. Since Inpp5eΔ/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 Inpp5eΔ/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 Inpp5eΔ/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.

Figure 4 with 4 supplements see all
Increased formation of layer V neurons in E18.5 Inpp5eΔ/Δ mutants.

(A–F) Coronal sections immunostained for the deep layer markers Tbr1 (layer VI) and Ctip2 (layer V) and for the upper layer marker Satb2 (layers II-IV); there is no obvious defect in layering in Inpp5eΔ/Δ embryos except for the formation of a heterotopia (asterisk in D). At caudal levels, the cortex becomes thinner and the rhinal fissure is shifted medially as indicated by the arrows. (G–O) Formation of cortical neurons at rostrolateral levels. The proportion of Tbr1+layer VI neurons is decreased with a concomitant increase in Ctip2+layer V neurons. (P–X) Portion of cortical neurons at rostromedial levels. Immunolabeling with cortical layer markers revealed no significant difference. Note that due to the thinner cortex, the position of layer VI Tbr1+ (Q) and layer V Ctip2+ neurons (J, S) appears to be shifted to more superficial positions; however, the relative order of these layers remains unaffected. All statistical data are presented as means ± 95% confidence intervals (CI); unpaired t-tests (M–O, X); Mann Whitney tests (V, W); n = 4; **p<0.01. Scale bars: 500 μm (A) and 100 μm (G). CC: corpus callosum; ctx: cortex; sep: septum; str: striatum.

A mutation in the ciliary gene Tctn2 leads to increased telencephalic neurogenesis

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 Inpp5eΔ/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 Tctn2Δ/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.

Increased generation of cortical neurons in the lateral neocortex of E12.5 Tctn2Δ/Δ embryos.

(A–C) Double immunofluorescence for PCNA and Tbr2 revealed a significantly decreased proportion of basal progenitors. (D–F) The portion of Tbr1+ cortical neurons was increased. All statistical data are presented as means ± 95% confidence intervals (CI); unpaired t tests; n = 4; **p<0.01; ***p<0.001. Scale bar: 50 μm. bv: blood vessel.

Ciliary defects in the forebrain of E12.5 Inpp5eΔ/Δ embryos

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 Inpp5eΔ/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 Inpp5eΔ/Δ neuroepithelial cells in the E12.5 telencephalon (Figure 6A,B) or diencephalon (data not shown).

Ciliary defects in E12.5 Inpp5eΔ/Δ forebrain.

(A–B) Immunohistochemistry for Arl13b and γ-Tubulin (γTUB) on E12.5 brain cryosections showed an accumulation of ciliary axonemes and basal bodies, respectively, at the apical border of radial glial cells facing the ventricules in both control (A) and Inpp5eΔ/Δ (B) embryos without any gross difference. Scale bars: 10 μm. (C–E) Scanning electron microscopy (SEM) on E12.5 control (C) and Inpp5eΔ/Δ (D, E) brains highlighted the presence of primary cilia projecting from the apical surface of radial glial cells in both control (A) and Inpp5eΔ/Δ (B) embryos. However, SEM also revealed the presence of abnormal cilia in Inpp5eΔ/Δ embryos having a spherical shape (arrows in D and E) or aberrant lateral buddings (arrowheads in D and E). Scale bars: 2 μm. (F–L) Transmission electron microscopy (TEM) analysis on E12.5 brains showed longitudinal sections of primary cilia in control (F) and Inpp5eΔ/Δ (G–L) embryos. In control primary cilia, the axoneme appeared as an extension of the basal body (bb, black arrowheards) (F–H). In addition to cilia with normal morphology, abnormal cilia were identified in Inpp5eΔ/Δ embryos thanks to the presence of a basal body apparently correctly docked to the apical membrane. Abnormal cilia lacked an axoneme (I, J, L) or showed unusual membranous structures, such as budding (G, K) or internal (I, K, L) vesicles (arrows) or undulating peripheral membranes (I). Note that tight junctions (white arrowheads in F and G) appeared normal in Inpp5eΔ/Δ (G) and control (F) embryos, suggesting that apico-basal polarity of Inpp5eΔ/Δradial glial cells was not compromised. Scale bars: 200 nm. (M–Q) TEM images showing transverse sections of the axoneme (M, O, P) and the basal body (N, Q) in control (M, N) and Inpp5eΔ/Δ (O–Q) embryos with no major difference in the basal bodies between control (N) and Inpp5eΔ/Δ (Q) embryos. However, transverse section of primary cilia in Inpp5eΔ/Δ brains revealed the presence of normal axonemes composed of nine correctly organized doublets of microtubules on some radial glial cells (O), while others harbored an abnormal axoneme containing a lower number of microtubule doublets (P). Scale bars: 50 nm. (R) Graph showing the number of normal versus abnormal cilia (cil.) found on TEM images from control (n = 3) or Inpp5eΔ/Δ (n = 3) embryos. cc: counted cilia.

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 Inpp5eΔ/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 Inpp5eΔ/Δ embryos. Cilia were recognized by basal bodies anchored to the apical membrane in both control and Inpp5eΔ/ΔRGCs (Figure 6F–L,N,Q). However, in Inpp5eΔ/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 Inpp5eΔ/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 Inpp5eΔ/Δ embryos, and found an increase in abnormal cilia in Inpp5eΔ/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 Inpp5eΔ/Δ embryos. These abnormalities are consistent with a role of Inpp5e in maintaining cilia stability (Jacoby et al., 2009).

Restoring Gli3 repressor ratio rescues cortical malformations in Inpp5eΔ/Δ embryos

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 Inpp5eΔ/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 Inpp5eΔ/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.

Figure 7 with 3 supplements see all
Re-introducing a single copy of the Gli3 repressor rescues the neurogenesis defect in E12.5 Inpp5e mutants.

(A–D) Gli3 western blot on E12.5 dorsal telencephalic tissue revealed the Gli3 full length (FL) and repressor (R) forms (A). While Gli3FL levels are not affected (B), levels of Gli3R (C) and the Gli3R/Gli3FL ratio (D) are decreased in Inpp5eΔ/Δ embryos. An unpaired t-test was used to evaluate levels of Gli3FL and Gli3R and the Gli3R/Gli3FL ratio in four control and fourInpp5eΔ/Δ embryos derived from four different litters. (E–L) Formation of basal progenitors and neurons in the neocortex of Inpp5eΔ/Δ and Inpp5eΔ/Δ;Gli3Δ699/+ embryos. In the lateral neocortex of E12.5 embryos, there is no significant difference in the proportions of Tbr1+ neurons (E, G, I, K) and basal progenitor cells (F, H, J, L) between control and Inpp5eΔ/Δ;Gli3Δ699/+ embryos. Note the three bulges of the ventral telencephalon in Inpp5eΔ/Δ;Gli3Δ699/+ embryos (J). Boxes indicate the regions where cell counts were performed. All statistical data are presented as means ± 95% confidence intervals (CI); unpaired t-tests (n = 5) (B–D) and one-way ANOVA followed by Tukey’s multiple comparison test (K, L); *p<0.05; **p<0.01; ***p<0.001. Scale bars: 250 μm (E), and 50 μm (E’). bv: blood vessel; ctx: cortex.

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 Gli3Δ699/+ mice that can only produce Gli3R in a cilia-independent manner (Besse et al., 2011; Bose, 2002). Overall inspection of Inpp5eΔ/Δ;Gli3Δ699/+ embryos revealed restored eye formation, whereas Inpp5eΔ/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 Inpp5eΔ/Δ;Gli3Δ699/+ embryos as compared to Inpp5eΔ/Δ embryos. In E18.5 Inpp5eΔ/ΔGli3Δ699/+ mutants, the corpus callosum has a thickness indistinguishable from that of control embryos (Figure 7—figure supplement 3). In E12.5 and E14.5 Inpp5eΔ/Δ;Gli3Δ699/+ embryos, the neocortex lacks the undulations of the VZ present in Inpp5eΔ/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 Inpp5eΔ/Δ embryos. While these changes were still present in Inpp5eΔ/Δ littermate embryos, there was no statistically significant difference between control and Inpp5eΔ/Δ;Gli3Δ699/+ embryos (Figure 7E–L). This finding indicates that the neurogenesis phenotype of E12.5 Inpp5eΔ/Δ mutants is rescued by a single copy of the Gli3Δ699 allele. We next investigated the formation of basal progenitors and of cortical projection neurons in E14.5 Inpp5eΔ/Δ;Gli3Δ699/+ embryos. The proportion of Tbr1+Ctip2+ neurons was not affected in the medial neocortex of E14.5 Inpp5eΔ/Δ;Gli3Δ699/+ embryos. In contrast, the proportion of Tbr1-Ctip2+ neurons was reduced as in Inpp5eΔ/Δ mutants (Figure 8A,C,E,I,J). Similarly, the proportions of basal progenitors in the medial Inpp5eΔ/ΔGli3Δ699/+ neocortex was slightly improved compared to Inpp5eΔ/Δ embryos but significantly smaller than in control embryos (Figure 8B,D,F,K). As re-introducing a single Gli3Δ699 allele does not completely rescue the Inpp5eΔ/Δ neurogenesis phenotype, we generated Inpp5eΔ/Δ embryos homozygous for the Gli3Δ699 allele. Interestingly, the morphology of the dorsal telencephalon including the hippocampal formation was indistinguishable between control and Inpp5eΔ/Δ;Gli3Δ699/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 Inpp5eΔ/Δ embryos, whereas two copies are required for a full rescue.

Two copies of the Gli3 repressor allele are required to rescue the neurogenesis defects in E14.5 Inpp5e mutants.

(A–H) Proportions of neurons (A, C, E, G) and basal progenitors (B D, F, H) in the medial neocortex of control, Inpp5eΔ/Δ, Inpp5eΔ/Δ;Gli3Δ699/+ and Inpp5eΔ/Δ;Gli3Δ699/Δ699 embryos. (A, C, E, G, J) The formation of Tbr1-Ctip2+ projection neurons is rescued after re-introducing two copies of the Gli3 repressor allele. (B, D, F, H, K) The proportion of basal progenitors is slightly increased in Inpp5eΔ/Δ;Gli3Δ699/+ embryos but a full rescue is only achieved in Inpp5eΔ/Δ;Gli3Δ699/Δ699 embryos. Boxes indicate the regions where cell counts were performed. All statistical data are presented as means ± 95% confidence intervals (CI); one-way ANOVA followed by Tukey’s multiple comparison test (I, J, K); *p<0.05; ***p<0.001. Scale bars: 250 μm (A, B), 50 μm (A’, B’). bv: blood vessel; ctx: cortex.

Discussion

Generating a functional cerebral cortex requires a finely tuned balance between direct and indirect neurogenesis to form subtypes of cortical projection neurons in appropriate numbers. Here, we show that the ciliary mouse mutants Inpp5e and Tctn2 present with a transient increase in neurons forming directly from radial glia progenitors in the lateral neocortex at the expense of basal progenitor formation. This increase in neurogenesis results in augmented formation of Ctip2+ layer V neurons in the Inpp5e mutant cortex. Our studies also revealed that the Inpp5e mutation interfered with the stability of the RGC primary cilium and its signaling functions, leading to a reduction in the Gli3R levels. Since re-introducing Gli3R in an Inpp5e mutant background restored the decreased formation of normal proportions of basal progenitors and neurons, our findings implicate a novel role for primary cilia in controlling the signaling events that direct the decision of RGCs to undergo either direct or indirect neurogenesis.

Primary cilia affect the decision between direct and indirect neurogenesis

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.

Inpp5e controls direct/indirect neurogenesis through Gli3 processing

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 Inpp5eΔ/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 Inpp5eΔ/Δ embryos. This idea is consistent with the findings that (i) Gli3Δ699/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 Gli3Xt/Xt mutants are not rescued in Shh-/-/Gli3XtXt 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 Inpp5eΔ/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 pAktS473 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 Inpp5eΔ/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 Inpp5eΔ/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).

Model for Inpp5e’s role in controlling direct vs indirect neurogenesis in the developing cortex.

(A) A fine-tuned balance between direct and indirect neurogenesis is required to produce cortical neurons in appropriate numbers. The structure of a primary cilium and the ciliary localization of the Inpp5e protein are schematically indicated. (B) The Inpp5e mutation affects the axoneme (shaded microtubules) and ciliary morphology and may compromise the transition zone as indicated by the grayish colour (Dyson et al., 2017). Gli3R levels are reduced and there is a shift towarddirect neurogenesis. (C) Tctn2Δ/Δ embryos have morphologically abnormal cilia, a defective axoneme and transition zone (Garcia-Gonzalo et al., 2011), lack ciliary Inpp5e protein (Garcia-Gonzalo et al., 2015) and phenocopy the neurogenesis defect of Inpp5eΔ/Δ mutants. (D) Introducing Gli3R in an Inpp5e mutant background restores Gli3 levels and the balance between direct and indirect neurogenesis. BB: basal body; BP: basal progenitor; PC: primary cilium; RGC: radial glial cell; TZ: transition zone.

Implications for Joubert syndrome

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 Inpp5eΔ/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 Inpp5eΔ/Δ mutant recapitulates cortical abnormalities in JS patients and starts to help unravelling the pathomechanisms underlying these defects.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent (Mus musculus)Inpp5edelta
(Inpp5etm1.2Sch)
PMID:19668215MGI:4360187
Genetic reagent (Mus musculus)Gli3delta699
(Gli3tm1Urt)
PMID:11978771MGI:2182576
Genetic reagent (Mus musculus)Tctn2delta
(Tctn2tm1.1Reit)
PMID:21725307MGI:5292130
AntibodyAnti-Arl13b
(clone N295B/66)
(Mouse monoclonal)
UC Davis/NIH NeuroMab FacilityCat# 75–287
RRID:AB_11000053
IF (1:1500)
AntibodyAnti-BrdU (Rat monoclonal)AbcamCat# ab6326
RRID:AB_305426
IF (1:50)
AntibodyAnti-BrdU/IdU (B44) (Mouse monoclonal)BD BiosciencesCat# 347580
RRID:AB_2313824
IF (1:500)
AntibodyCleaved-Caspase3 (Asp175) (5A1E) (Rabbit polyclonal)Cell Signaling TechnologyCat# 9664
RRID:AB_2070042
IF (1:100)
AntibodyAnti-Ctip2 (Rat monoclonal)AbcamCat# ab18465 RRID:AB_2064130IF (1:1000)
AntibodyAnti-GFAP (Rabbit polyclonal)AgilentCat# Z0334 RRID:AB_10013382IF (1:1000)
AntibodyAnti-L1, clone 324 (Rat monoclonal)MilliporeCat# MAB5272 RRID:AB_2133200IF (1:1000)
AntibodyAnti-Pax6 (Rabbit polyclonal)BiolegendCat# 901301
RRID:AB_2565003
IF (1:400)
AntibodyAnti-PCNA (PC10) (Mouse monoclonal)AbcamCat# ab29 RRID:AB_303394IF (1:500)
AntibodyAnti-Prox1 (Rabbit polyclonal)ReliatechCat# 102-PA32
RRID:AB_10013821
IHC (1:1000)
AntibodyAnti-pHH3 (Rabbit polyclonal)MilliporeCat# 06–570 RRID:AB_310177IF (1:100)
AntibodyAnti-Satb2 (Mouse monoclonal)AbcamCat# ab51502
RRID:AB_882455
IF (1:200)
AntibodyAnti-Tbr1 (Rabbit polyclonal)AbcamCat# ab31940 RRID:AB_2200219IF (1:400)
AntibodyAnti-Tbr2 (Rabbit polyclonal)AbcamCat# ab23345 RRID:AB_778267IF (1:1000)
AntibodyAnti−γTUB, (Rabbit polyclonal)Sigma AldrichCat# SAB4503045 RRID:AB_10747615
IF (1:100)
AntibodyAnti-mouse Cy2 secondary (Donkey polyclonal)Jackson ImmunoResearch LabsCat# 715-225-151 RRID:AB_2340827IF (1:100)
AntibodyAnti-rabbit Cy3 secondary (Donkey polyclonal)Jackson ImmunoResearch LabsCat# 711-165-152IF (1:100)
AntibodyAnti-rat Cy3 secondary (Goat polyclonal)Jackson ImmunoResearch LabsCat# 711-165-152 RRID:AB_2307443IF (1:100)
AntibodyAnti-rabbit Alexa Fluor 488 secondary (Goat polyclonal)Molecular Probes (now: Invitrogen)Cat# A-11008 RRID:AB_143165IF (1:200)
AntibodyAnti-rat Alexa Fluor 647 secondary (Goat polyclonal)Molecular Probes
(now: Invitrogen)
Cat# A-21247 RRID:AB_141778IF (1:200)
AntibodyBiotinylated swine anti-rabbit IgGDakoCat# E0431IF (1:400)
AntibodyStreptavidin, Alexa Fluor 488 conjugate antibodyMolecular Probes
(now: Invitrogen)
Cat# S32354 RRID:AB_2315383IF (1:100)
AntibodyStreptavidin, Alexa Fluor 568 conjugate antibodyThermo Fisher ScientificCat# S-11226 RRID:AB_2315774IF (1:100)
AntibodyBiotinylated goat anti-rabbit IgGDako
(now: Agilent)
Cat# E0432
RRID:AB_2313609
IF (1:400)
AntibodyAnti-h/m Gli3 (Goat polyclonal)R and D SystemsCat# AF3690 RRID:AB_2232499WB (1:500)
AntibodyAnti-β-Actin (clone AC-15) (Mouse monoclonal)AbcamCat# ab6276 RRID:AB_2223210WB (1:15,000)
AntibodyIRDye 680RD Donkey anti-Goat IgGLI-COR BiosciencesCat# 926–68074
RRID:AB_10956736
WB (1:15,000)
AntibodyIRDye 800CW Donkey anti-Mouse IgGLI-COR BiosciencesCat# 925–32212
RRID:AB_2716622
WB (1:15,000)
Commercial assay or kitVECTASTAIN Elite ABC-Peroxidase Kit
Vector LaboratoriesCat# PK-6100
RRID:AB_2336819
Chemical compound, drugIdU 5-Iodo-2′-deoxyuridineSigma AldrichCat# I7125(10 mg/ml)
Chemical compound, drugBrdU 5-Bromo-2′-deoxyuridineSigma AldrichCat# B5002(10 mg/ml)
Software, algorithmFijiPMID:22743772?PRID:SCR_002285http://imagej.net/Fiji
Software, algorithmImage Studio LiteLi-Cor4.0
Software, algorithmGraphPad PrismGraphPad Software8.4.2 (679)
Software, algorithmAdobe PhotoshopAdobe Inc12.1
OtherDAPI
(4',6-Diamidino-2-Phenylindole, Dihydrochloride)
Thermo Fisher ScientificCat# D1306
RRID:AB_2629482
IF (1:2000)

Mice

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. Inpp5eΔ (Inpp5edelta), Gli3Δ699 (Gli3delta699) and Tctn2Δ (Tctn2tm1.1Reit) mouse lines have been described previously (Bose, 2002; Garcia-Gonzalo et al., 2011; Jacoby et al., 2009). Inpp5eΔ/+ mice were interbred to generate Inpp5eΔ/Δ embryos; exencephalic Inpp5eΔ/Δ embryos which made up ca. 25% of homozygous mutant embryos were excluded from the analyses. Wild-type and Inpp5eΔ/+ litter mate embryos served as controls. Inpp5eΔ/Δ;Gli3Δ699/+ and Inpp5eΔ/Δ;Gli3Δ699/Δ699 embryos were obtained from inter-crosses of Inpp5eΔ/+;Gli3Δ699/+ mice using wild-type, Inpp5eΔ/+ and Gli3Δ699/+ 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.

Immunohistochemistry and in situ hybridization

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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).

Western blot

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Protein was extracted from the dorsal telencephalon of E12.5 wild-type and Inpp5eΔ/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.

Scanning and transmission electron microscopy

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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.

Statistical analyses

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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.

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Decision letter

  1. Fadel Tissir
    Reviewing Editor; Universite' Catholique de Louvain, Belgium
  2. Marianne E Bronner
    Senior Editor; California Institute of Technology, United States
  3. Fadel Tissir
    Reviewer; Universite' Catholique de Louvain, Belgium

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This study focuses on the role of Inpp5e, a protein essential for the integrity of primary cilia, in the development of the cerebral cortex. The authors find that mice mutant for Inpp5e have severe morphological defects in the primary cilium of cortical apical Radial Glia Cells, and this is linked to changes in the mode of neurogenesis, favoring direct neurogenesis against indirect neurogenesis via Intermediate Progenitor Cells (IPCs). This effect is nicely shown by studying the abundance of cells positive for marker proteins of IPCs and newborn neurons, as well as cell cycle exit. Intriguingly, this defect is transient during early stages of neurogenesis (circa E12.5), not observable at later stages, but has lasting effects on the abundance of neurons in deep cortical layers. Part of the same defects are found in mouse embryos mutant for Tctn2, another ciliary protein. Then the authors go on to investigate the signaling cascades affected in this mutant and leading to the changes in mode of neurogenesis, and find no changes in Erk but a significant increase in mTOR signaling. Finally, the authors focus on Shh signaling, a well known pathway tightly linked to the primary cilium. They find that the neurogenesis phenotype relates to impaired Gli3 processing and a loss of its repressive form Gli3R. Finally, the authors analyze Gli3D699 mutant mice, where Gli3R is produced independently from the primary cilium, in an Inpp5e mutant background, to show a dose-dependent rescue of the cortical phenotypes, and thus demonstrating that Inpp5e regulates the modes of cortical neurogenesis by regulating Gli3R levels.

Decision letter after peer review:

Thank you for sending your article entitled "A transient role of primary cilia in controlling direct versus indirect neurogenesis in the developing cerebral cortex" for peer review at eLife. Your article is being evaluated by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation is being overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor.

Given the list of essential revisions, including new experiments, the editors and reviewers invite you to respond as soon as you can with an action plan for the completion of the additional work. We expect a revision plan that under normal circumstances can be accomplished within two months, although we understand that in reality revisions will take longer at the moment. We plan to share your responses with the reviewers and then advise further with a formal decision.

Summary:

This study focuses on the role of Inpp5e, a protein essential for the integrity of primary cilia, in the development of the cerebral cortex. The authors find that mice mutant for Inpp5e have severe morphological defects in the primary cilium of cortical apical Radial Glia Cells, and this is linked to changes in the mode of neurogenesis, favoring direct neurogenesis against indirect neurogenesis via Intermediate Progenitor Cells (IPCs). This effect is nicely shown by studying the abundance of cells positive for marker proteins of IPCs and newborn neurons, as well as cell cycle exit. Intriguingly, this defect is transient during early stages of neurogenesis (circa E12.5), not observable at later stages, but has lasting effects on the abundance of neurons in deep cortical layers. Part of the same defects are found in mouse embryos mutant for Tctn2, another ciliary protein. Then the authors go on to investigate the signaling cascades affected in this mutant and leading to the changes in mode of neurogenesis, and find no changes in Erk but a significant increase in mTOR signaling. Finally, the authors focus on Shh signaling, a well known pathway tightly linked to the primary cilium. They find that the neurogenesis phenotype relates to impaired Gli3 processing and a loss of its repressive form Gli3R. Finally, the authors analyze Gli3D699 mutant mice, where Gli3R is produced independently from the primary cilium, in an Inpp5e mutant background, to show a dose-dependent rescue of the cortical phenotypes, and thus demonstrating that Inpp5e regulates the modes of cortical neurogenesis by regulating Gli3R levels.

The study is nicely planned and performed. However, there are points that must be addressed before the manuscript is ready for publication. In particular:

1) Important clarifications need to be made with regards to the neurogenesis data. Images and illustrations should be reflective of the phenotypes described in the text, and a better discussion is required to reconcile conflicting data.

2) The mTOR/ERK part lacks key controls and is not central to the message of the paper. The section should be removed.

Essential revisions:

1) Signs of cortical distortions, suggestive of patterning defects are seen at the rostral part of the cortex (Figure 1B, F, G). Pax6 expression pattern (gradient) seems also affected in the mutant (decreased Pax6 medially). Please show a more representative image or explain/comment in the legend.

2) Subsection “Inpp5e controls direct vs indirect neurogenesis in the lateral neocortex”: The authors state: "these findings show that initially Tbr1+ and later Ctip2+Tbr1- neurons were increasingly formed in the lateral neocortex of Inpp5e embryos". Is that specific to the mutant? It is also the case for control embryos but maybe at a different time point. Please rephrase the sentence.

3) The authors claim that there is an increased neuron production. This does not seem to be the case from Figure 2I, J.

4) Difficult to reconcile results of Figure 2I, J and Figure 3. Please comment and discuss.

5) How can authors conclude that there is no change in the size of the primary cilium from IHC Figure 6 A, B and subsection “Ciliary defects in the forebrain of E12.5 Inpp5eΔ/Δ embryos”? This is contradictory to SEM results Figure 6 C-E. Is IHC the best way to assess the size of cilia?

6) The patterning defect (see Supplementary Figure 10 C, D, G H) is such that it makes the results of neurogenesis difficult to interpret.

7) The abundance of IPCs is analyzed by stains against Tbr2/PCNA, and PH3 mitoses at basal position. A decrease in both is interpreted as a loss of IPCs, but in fact the authors find conflicting results in their mutant: loss of Tbr2+PCNA+ cells in the lateral cortex but not medial (Figure 1), and then loss of basal PH3 in medial cortex but not lateral (Figure 3—figure supplement 2).

8) Quantification of PCNA+ cells shows changes in the proportion that are Tbr2+. Importantly, 80% of PCNA+ were Pax6+, and 40% were Tbr2+, so a minimum of 20% of PCNA+ cells are co-expressing Pax6 and Tbr2. Contrary to these results, existing evidence in the field shows that the overlap in Pax6 and Tbr2 expression is really residual in mouse. Please comment on that.

9) Which neuron subpopulations are identified as Ctip2+/Tbr1+ and Ctip2+/Tbr1-?

10) Why increased direct neurogenesis diminishes the overall production of Tbr1 neurons (though higher at E12.5), but with no changes in intermediate and upper layer neurons? This is opposite from what was shown by Cardenas and colleagues in 2018, where forced direct neurogenesis in the early NCx increases deep layer neurons and diminishes upper layer neurons.

11) Subsection “Cortical malformations in Inpp5eΔ / Δ embryos” – "This analysis showed that the mutant cortex was thinner laterally but not medially with a more pronounced reduction of the thickness at caudal levels (Figure 4—figure supplement 2)." The data shown in Figure 4—figure supplement 2 contradict this description. In addition, DAPI images are very dark with any detail very difficult to see. These must be better visible, for example by showing in black and white.

12) For the entire Figure 7, pictures and quantification of all variables must include the results from simple lnpp5e mutants. Although these results are shown in earlier figures, they are key to assess the rescue effects of Gli3D699 in hetero and homozygosity, and so they must be presented again in this figure. One clear example of the importance of showing these results here is the phenotype shown (but not mentioned) in Figure 7I, where the dorsal cortex seems to be much shorter than in the control embryo (see the lateral end of the Tbr2+ region, inset box), plus there seem to be three basal ganglia. This is not mentioned as part of the phenotype in lnpp5e mutants. It this caused by this compound genotype?

13) Similar to the previous point, the analysis of Gli3 rescue must include littermates mutant only for, to show if the heterozygous expression of Gli3D699 rescues (or worsens) the deficit in Tbr1+Ctip2+, Tbr1-Ctip2+ and Tbr2+ cells in lnpp5e mutants.

14) Figure 7, panels Q and Q' are at different magnification than the rest of the figure. Pictures of Tbr1 and Ctip2 stains are at insufficient magnification to illustrate the dramatic differences described in the manuscript and quantified in panels P and S.

15) There is a part of the study focused on potential effects in Erk and mTOR signaling. While potentially interesting, this part is completely disconnected from the rest of the study (in fact all the data is in Supplementary Figures), and the authors decide to ignore in the rest of the manuscript, instead focus on Shh signaling. Thus, Erk and mTOR signaling analyses should be removed.

16) Given that other primary cilia mutants do not exhibit deficits in direct versus indirect neurogenesis, as discussed by the authors, they must change the title of their manuscript to indicate that it is Inpp5e that controls this process. Indeed, as also discussed by the authors, this protein plays multiple other roles in brain development (eye, hippocampus, corpus callosum), and thus it is likely to play other roles in cortical development that may contribute to regulate the mode and rate of neurogenesis, independent from the primary cilium.

17) Figure 1—figure supplement 1B,E – PSPB is not visible in KOs, and impossible to assess the existence of a phenotype in this area. These images must be re-framed to make this visible.

18) In Figure 1—figure supplement 3, the authors show some folds in VZ of NCx and hippocampal anlage. Whereas this is not the core of the main findings, the authors must disclose the penetrance and severity of this phenotype.

19) Throughout the manuscript, the authors need to provide the actual numbers for the means in each of their graphs. This could be in the text or figure legend, but in the current version, this data is not present.

20) Could the authors please provide higher magnification images for Figure 1A-J, Figure 2A-F?

21) Figure 3: could the authors indicate that this data is from lateral cortex in the actual figure?

22) In several figures, the cortex is clearly thinner (as the authors indicate in Figure 4—figure supplement 2). However, this seems to be much thinner than would be accounted for by a relatively small decrease in basal progenitor production. Apical progenitor production (S4) and progenitor cell cycle (S5) appears unaffected. Did the authors examine whether there was increased cell death or either progenitors or postmitotic neurons in the mutants?

23) In Figure 4P-U, the cortical layers from the controls and mutants are not aligned. Is this because the overall cortex is thinner and the images are aligned from the basal surface? Including DAPI for these sections would help here.

24) Is the data in Figure 5 from medial or lateral cortex?

25) Why did the authors switch to a parametric t-test for the western blots in Figure 7, S10, S11? Shouldn't a paired non-parametric test (ie: paired samples Wilcoxon) be used here? Also, the data should not be displayed as paired with the lines joining the control and KO samples.

26) For the experiments in S11, the authors show that pAkt/pS6 are elevated Inpp5e-/- mutants, consistent with increased mTOR activity. They attempt to rule this pathway out by showing that treatment with rapamycin for 24hours at E11.5 does not affect the basal progenitor phenotype in Inpp5e-/- mutants. However, the authors do not provide any data showing the effectiveness of their rapamycin treatment (ie, normalization of pAkt/pS6 to wildtype levels). I think they need to be very careful about their interpretation of these results without additional controls.

27) Do the authors have an explanation as to why the phenotypes are predominantly in lateral cortex at E12.5 and medial cortex at E14.5? This seems like something that should be mentioned in the discussion.

28) A model figure at the end would be very useful for explaining how loss of Inppe5 leads to reduced Gli3R levels and alterations in basal progenitor production.

https://doi.org/10.7554/eLife.58162.sa1

Author response

Essential revisions:

1) Signs of cortical distortions, suggestive of patterning defects are seen at the rostral part of the cortex (Figure 1B, F, G). Pax6 expression pattern (gradient) seems also affected in the mutant (decreased Pax6 medially). Please show a more representative image or explain/comment in the legend.

In the original manuscript, we very carefully described patterning defects before we start analysing cortical stem cell development. In brief, the hippocampal anlage is the most affected structure, probably due to reduced Wnt gene expression in the cortical hem, but this structure is not the focus of this study. The neocortex itself is undulated at E12.5 but has a smooth surface by E14.5 and shows a mild patterning defect at the pallial/subpallial boundary. It expresses the progenitor markers Emx1, Lhx2, Pax6 and Ngn2 with reduced Pax6 expression levels in the medial neocortex. We think that these mild patterning defects do not invalidate our analysis and that Figure 1B, F and J are representative of the Inpp5e mutant telencephalon.

2) Subsection “Inpp5e controls direct vs indirect neurogenesis in the lateral neocortex”: The authors state: "these findings show that initially Tbr1+ and later Ctip2+Tbr1- neurons were increasingly formed in the lateral neocortex of Inpp5e embryos". Is that specific to the mutant? It is also the case for control embryos but maybe at a different time point. Please rephrase the sentence.

We rephrased the sentence making it clearer that in the lateral neocortex of Inpp5eΔ / Δ embryos we observed an increase in the formation of Tbr1+ neurons at E12.5 followed by an augmented generation of Ctip2+Tbr1- neurons at E14.5 (subsection “Inpp5e controls direct vs indirect neurogenesis in the lateral neocortex”).

3) The authors claim that there is an increased neuron production. This does not seem to be the case from Figure 2I, J.

In our analysis of neuron formation at E14.5 (Figure 2I-N) we distinguish between two neuronal subpopulations, a Tbr1+/Ctip2+ and a Tbr1-/Ctip2+ population. We show that there is only an increase in the proportion of Tbr1-/ Ctip2+ population in the lateral neocortex which can be clearly seen in the high magnification insets (Figure 2I’, I’’, J’ and J’’). This might not be so obvious from the overview picture (Figure 2I and J) but one needs to consider that the mutant cortex is thinner and we counted cell proportions rather than absolute neuron numbers. To make our point clearer, we used arrows and arrowheads to indicate the different neuron populations in Figure 2I’, I’’, J’ and J’’. We also re-worded the sentence to make it clearer that proportions of neurons are affected (subsection “Inpp5e controls direct vs indirect neurogenesis in the lateral neocortex”).

4) Difficult to reconcile results of Figure 2I, J and Figure 3. Please comment and discuss.

We think there is a misunderstanding. Figure 2I, J show the proportion of Tbr1+ and Ctip2+ neurons in E14.5 embryos while Figure 3 shows a cell cycle exit experiment in E12.5 embryos. Due to the different ages and types of experiments, these two figures are not directly comparable.

5) How can authors conclude that there is no change in the size of the primary cilium from IHC Figure 6 A, B and subsection “Ciliary defects in the forebrain of E12.5 Inpp5eΔ/Δ embryos”? This is contradictory to SEM results Figure 6 C-E. Is IHC the best way to assess the size of cilia?

We agree with the reviewer that immunofluorescence does not allow us to measure the size of cilia and we have removed this formulation from the revised manuscript (subsection “Ciliary defects in the forebrain of E12.5 Inpp5eΔ / Δ embryos”).

6) The patterning defect (see Supplementary Figure 10 C, D, G H) is such that it makes the results of neurogenesis difficult to interpret.

This comment is very similar to point #1. As outlined above, we have carefully analysed patterning in the Inpp5e mutant and think that the mild patterning defects do not interfere with our conclusion on neurogenesis defects in the neocortex. To this specific figure: in the dorsal telencephalon, the most obvious morphological difference in Figure 10 C, D, G, H is a malformation of the hippocampus which is not the focus of this paper. At the rostrocaudal levels we have been investigating the mutant, the neocortex is undulated at E12.5 but smoothens at E14.5 and is otherwise not severely affected. We think that this undulation does not prevent us from investigating the process of direct neurogenesis and the underlying mechanisms. Finally, this figure represents our analysis of Erk signalling but we have removed this analysis from the manuscript as suggested by other reviewers (see point 15).

7) The abundance of IPCs is analyzed by stains against Tbr2/PCNA, and PH3 mitoses at basal position. A decrease in both is interpreted as a loss of IPCs, but in fact the authors find conflicting results in their mutant: loss of Tbr2+PCNA+ cells in the lateral cortex but not medial (Figure 1), and then loss of basal PH3 in medial cortex but not lateral (Figure 4—figure supplement 2).

Tbr2 and pHH3 antibodies label different groups of cells. Tbr2 labels all basal progenitors whereas pHH3 staining identifies mitotic basal progenitors. This different specificity makes it difficult to directly compare the two results but we interpret this finding that the E12.5 basal progenitors in the medial neocortex may have reduced proliferation rates. Indeed, the Inpp5e mutants show a reduction in the proportion of basal progenitors in the E14.5 medial neocortex and this reduction may at least partially be explained by the earlier lower proliferation rate. We included this consideration in subsection “Inpp5e controls direct vs indirect neurogenesis in the lateral neocortex” of the revised manuscript.

Finally, the reviewer alludes to different effects of the Inpp5e mutation on medial and lateral neocortex. We think there are several possible explanations. The phenotypes may follow the neurogenesis gradient with lateral parts of the neocortex being more advanced in basal progenitor and neuron formation. There is also a reduced Pax6 expression in the medial neocortex that is likely to affect neurogenesis. We have included this consideration in the Discussion section.

8) Quantification of PCNA+ cells shows changes in the proportion that are Tbr2+. Importantly, 80% of PCNA+ were Pax6+, and 40% were Tbr2+, so a minimum of 20% of PCNA+ cells are co-expressing Pax6 and Tbr2. Contrary to these results, existing evidence in the field shows that the overlap in Pax6 and Tbr2 expression is really residual in mouse. Please comment on that.

Englund et al., reported that 11.5 +/- 1% of cells co-express Pax6 and Tbr2 in the E14.5 neocortex (Englund et al., 2005), i.e. not too far off the number the reviewer estimated from our Pax6/PCNA and Tbr2/PCNA staining. However, this estimate is derived indirectly from two separate stains and hence might not easily be comparable to a Pax6/Tbr2 double stain.

9) Which neuron subpopulations are identified as Ctip2+/Tbr1+ and Ctip2+/Tbr1-?

We observed that in the E12.5 cortical plate almost all neurons co-express Ctip2 and Tbr1. This population is still present at E14.5 but there is an additional group of neurons which express Ctip2 only, i.e. are Tbr1-/ Ctip2+. Based on the time of their appearance the Tbr1+/Ctip2+ and Tbr1-/Ctip2+ neurons are likely to correspond to layer VI and layer V neurons, respectively, but we do not have definite proof for this. As identifying the fate of these neurons is not the main purpose of this manuscript, we prefer not to go deeper into this issue.

10) Why increased direct neurogenesis diminishes the overall production of Tbr1 neurons (though higher at E12.5), but with no changes in intermediate and upper layer neurons? This is opposite from what was shown by Cardenas and colleagues in 2018, where forced direct neurogenesis in the early NCx increases deep layer neurons and diminishes upper layer neurons.

The reviewer is correct that there is no change in upper layer neuron formation despite the early increase in direct neurogenesis. In the original manuscript, we have already provided a couple of possible explanations. First, the number of basal progenitors has normalized by E14.5 when the majority of upper layer neurons are born. Secondly, there are feedback signals from newly born neurons to radial glial cells to control the sequential production of deep and upper layer neurons. In addition to Notch signalling as described in Cardenas, these signals include Fgf9 and Neurotrophin 3. Inpp5e could influence this signalling by controlling cilia stability and/or levels of PIP3 which acts as a second messenger in receptor tyrosine kinase signalling. We think these considerations address the reviewer’s concern.

11) Subsection “Cortical malformations in Inpp5eΔ / Δ embryos” – "This analysis showed that the mutant cortex was thinner laterally but not medially with a more pronounced reduction of the thickness at caudal levels (Figure 4—figure supplement 2)." The data shown in Figure 4—figure supplement 2 contradict this description. In addition, DAPI images are very dark with any detail very difficult to see. These must be better visible, for example by showing in black and white.

We are grateful for the reviewer to raise this point. We have amended the text to say that “most of the mutant cortex was thinner except for the rostrolateral level” (subsection “Cortical malformations in Inpp5eΔ / Δ embryos”). We have changed the colour of the DAPI staining to black and white (Figure 4—figure supplement 2).

12) For the entire Figure 7, pictures and quantification of all variables must include the results from simple lnpp5e mutants. Although these results are shown in earlier figures, they are key to assess the rescue effects of Gli3D699 in hetero and homozygosity, and so they must be presented again in this figure. One clear example of the importance of showing these results here is the phenotype shown (but not mentioned) in Figure 7I, where the dorsal cortex seems to be much shorter than in the control embryo (see the lateral end of the Tbr2+ region, inset box), plus there seem to be three basal ganglia. This is not mentioned as part of the phenotype in lnpp5e mutants. It this caused by this compound genotype?

In the revised manuscript, we included the analysis of Inpp5eΔ / Δ;Gli3+/+ littermates which we obtained from the Gli3 rescue crosses (Figure 7 and Figure 8). In these embryos, we observed a reduced proportion of basal progenitors and an increased fraction of cortical neurons as in Inpp5eΔ / Δ embryos derived from the mating of Inpp5e heterozygous animals. Moreover, our analysis revealed that the addition of a single Gli3Δ699 allele slightly improved the formation of basal progenitors in the E14.5 medial neocortex compared to Inpp5e∆/∆ embryos but the proportion of basal progenitors was still significantly smaller than in control embryos. These findings support our conclusion that restoring the Gli3 repressor ratio rescues cortical malformations in Inpp5eΔ / Δ embryos.

Although the neocortex of Inpp5eΔ / Δ ; Gli3 Δ699/+ embryos appears shorter, we can clearly identify the neocortex based on the Tbr1 and Tbr2 staining. At this stage, the three bulges in the ventral telencephalon are specific to and appeared in all E12.5 Inpp5e Δ/Δ;Gli3Δ699/+ embryos we analysed; we did not note those in Inpp5eΔ / Δ embryos. We will added this information to the legend for Figure 7.

13) Similar to the previous point, the analysis of Gli3 rescue must include littermates mutant only for, to show if the heterozygous expression of Gli3D699 rescues (or worsens) the deficit in Tbr1+Ctip2+, Tbr1-Ctip2+ and Tbr2+ cells in lnpp5e mutants.

As outlined for the previous point, we analysed Inpp5eΔ / Δ;Gli3+/+ littermate embryos. The addition of a single Gli3Δ699 allele rescued the formation of basal progenitors and Tbr1+ neurons in the lateral neocortex at E12.5 (Figure 7) and led to a slight improvement in the proportions basal progenitors in the E14.5 medial neocortex (Figure 8). The full restoration of basal progenitors and of Tbr1-Ctip2+ neurons in the E14.5 medial neocortex required two copies of the Gli3Δ699 allele (Figure 8).

We will perform all analyses and quantifications (Tbr1/Ctip2 and Tbr2/PCNA) on E12.5 and E14.5 Inpp5eΔ / Δ;Gli3+/+ littermates from the Gli3 rescue experiment as in Figure 7 of the original manuscript.

14) Figure 7, panels Q and Q' are at different magnification than the rest of the figure. Pictures of Tbr1 and Ctip2 stains are at insufficient magnification to illustrate the dramatic differences described in the manuscript and quantified in panels P and S.

We have increased the magnifications of the insets of what is Figure 8 A’, C’, E’ and G’ in the revised manuscript. This and addition of arrows pointing at Ctip2+/Tbr1- neurons should better illustrate the differences in this population. We also ensured that the panels from the different genotypes have the same magnification.

15) There is a part of the study focused on potential effects in Erk and mTOR signaling. While potentially interesting, this part is completely disconnected from the rest of the study (in fact all the data is in Supplementary Figures), and the authors decide to ignore in the rest of the manuscript, instead focus on Shh signaling. Thus, Erk and mTOR signaling analyses should be removed.

We have removed the Erk and mTOR analyses from the manuscript. We agree with the reviewers that the major focus of the manuscript is on Gli3 signalling and the manuscript makes better reading without the Erk and mTOR analyses which appear disconnected.

16) Given that other primary cilia mutants do not exhibit deficits in direct versus indirect neurogenesis, as discussed by the authors, they must change the title of their manuscript to indicate that it is Inpp5e that controls this process. Indeed, as also discussed by the authors, this protein plays multiple other roles in brain development (eye, hippocampus, corpus callosum), and thus it is likely to play other roles in cortical development that may contribute to regulate the mode and rate of neurogenesis, independent from the primary cilium.

We have changed the title and made a corresponding change at the end of the Introduction. Mutations in ciliary genes can have a variety of effects on cortical development (see Discussion section), however, we not only found an increase in direct neurogenesis in the Inpp5e mutant but also in the Tctn2 mutant. This suggests that Inpp5e‘s effect on direct neurogenesis is more general and might for example be obscured by more severe patterning defects in other ciliary mutants. Moreover, the other phenotypes mentioned by the reviewer (eye, hippocampus, corpus callosum) are also rescued by re-introducing Gli3R strongly suggesting that these phenotypes are also cilia dependent.

17) Figure 1—figure supplement 1B,E – PSPB is not visible in KOs, and impossible to assess the existence of a phenotype in this area. These images must be re-framed to make this visible.

We think there must be a misunderstanding. Figure 1—figure supplement 1B, E focusses on the formation of the corticoseptal boundary not the PSPB as indicated in the figure legend. PSPB formation is analysed in Figure 1—figure supplement 1G-L with higher magnification insets clearly showing scattered Pax6+ and Dlx2 expressing cells.

18) In Figure 1—figure supplement 3, the authors show some folds in VZ of NCx and hippocampal anlage. Whereas this is not the core of the main findings, the authors must disclose the penetrance and severity of this phenotype.

We observed these folds which occur with 100% penetrance only at caudal positions, i.e. at the rostral/caudal level of the thalamus. Moreover, they become more prominent at more caudal levels. We have added this information in the revised manuscript (subsection “Inpp5eΔ / Δ embryos show mild telencephalic patterning defects”) but we would like to emphasize that this interesting observation is not the main focus of the manuscript. Indeed, these folds complicate the analysis of neurogenesis defects in the caudal neocortex. For this reason, we have not included this region in our analysis.

19) Throughout the manuscript, the authors need to provide the actual numbers for the means in each of their graphs. This could be in the text or figure legend, but in the current version, this data is not present.

In the originally submitted manuscript, we included an Excel file (Supplementary file 1) providing a summary of descriptive statistics of all tests used in this manuscript. We prefer this approach as it increases the readability of the main text but also provides the interested reader with much more information on statistical test results than we could include in the main text. We should have referred to this table in the original manuscript but have done this in the revised version at the end of the statistics paragraph (Materials and methods section). We will, however, follow the reviewer’s advice to include mean numbers in the main text if they prefer us to do so.

20) Could the authors please provide higher magnification images for Figure 1A-J, Figure 2A-F?

In the revised manuscript we included representative higher magnification images for Figure 1 and Figure 2.

21) Figure 3: could the authors indicate that this data is from lateral cortex in the actual figure?

We can confirm that this data is from lateral cortex and we have amended the figure legend correspondingly.

22) In several figures, the cortex is clearly thinner (as the authors indicate in Figure 4—figure supplement 2). However, this seems to be much thinner than would be accounted for by a relatively small decrease in basal progenitor production. Apical progenitor production (S4) and progenitor cell cycle (S5) appears unaffected. Did the authors examine whether there was increased cell death or either progenitors or postmitotic neurons in the mutants?

We have examined cell death but found very few apoptotic cells in the cortex of both, control and mutant embryos. This new data is included in Figure 3—figure supplement 1.

23) In Figure 4P-U, the cortical layers from the controls and mutants are not aligned. Is this because the overall cortex is thinner and the images are aligned from the basal surface? Including DAPI for these sections would help here.

The reviewer is correct, the overall cortex is thinner at caudal levels (for quantification see Figure 4—figure supplement 2) and images in Figure 4P-U are aligned from the basal surface. We included this information already in the figure legend of the original manuscript. Unfortunately, the high power pictures were taken without the DAPI channel.

24) Is the data in Figure 5 from medial or lateral cortex?

We can confirm that this data is from the lateral cortex, this information was added to the figure legend.

25) Why did the authors switch to a parametric t-test for the western blots in Figure 7, S10, S11? Shouldn't a paired non-parametric test (ie: paired samples Wilcoxon) be used here? Also, the data should not be displayed as paired with the lines joining the control and KO samples.

We realized that the background normalisation of the Western blot using an automatic function of the LI-COR software was not adequate. The protein lysates are derived from single dorsal telencephali and therefore have a low protein concentration. As a consequence, we needed to load large volumes of the lysates which might have caused some smiling, especially for the Gli3R bands. To take this effect into account, we repeated the background normalisation by placing a user defined box into each lane. The original blot and how we evaluated the blot is shown in Author response image 1:

Author response image 1

Testing the new data set for normality (Shapiro-Wilk test) and for equal variance revealed that the data is normally distributed and that there is no significant difference in variance between the control and mutant data sets. For this reason, we used an unpaired t-test to statistically evaluate this analysis.To use the above approach consistently throughout the paper, we tested all our data sets for normal distribution and equal variance. To evaluate statistical significance, we used unpaired t-tests for normally distributed data showing equal variance and non-parametric tests otherwise. In this way, we employed statistical tests consistently throughout the paper. This procedure is added to the Material and methods section. We would like to emphasize that this did not change the outcomes except for a significantly reduced proportion of mitotic RGCs in the E12.5 medial neocortex (Figure 3—figure supplement 2A, C). which does not, however, affect our conclusions. Moreover, we have summarized all details of the statistical tests in Supplementary file 1.

26) For the experiments in S11, the authors show that pAkt/pS6 are elevated Inpp5e-/- mutants, consistent with increased mTOR activity. They attempt to rule this pathway out by showing that treatment with rapamycin for 24 hours at E11.5 does not affect the basal progenitor phenotype in Inpp5e-/- mutants. However, the authors do not provide any data showing the effectiveness of their rapamycin treatment (ie, normalization of pAkt/pS6 to wildtype levels). I think they need to be very careful about their interpretation of these results without additional controls.

As suggested in the editor’s comments, we have removed the analysis of Akt and mTOR signalling in the revised manuscript.

27) Do the authors have an explanation as to why the phenotypes are predominantly in lateral cortex at E12.5 and medial cortex at E14.5? This seems like something that should be mentioned in the discussion.

A similar issue was already raised under point #7 and, as outlined in more detail in our response to this point, this differential effect may be due to the lateral to medial gradient of neurogenesis and/or gene expression changes ( for example Pax6). We have included this consideration in the Discussion section.

28) A model figure at the end would be very useful for explaining how loss of Inppe5 leads to reduced Gli3R levels and alterations in basal progenitor production.

We have included a model (Figure 9) summarizing our findings on the different mutants used in this manuscript. This new figure supports the discussion in subsection “Inpp5e controls direct/indirect neurogenesis through Gli3 processing” of how loss of Inpp5e functions leads to reduced Gli3 R levels and changes in direct and indirect neurogenesis.

https://doi.org/10.7554/eLife.58162.sa2

Article and author information

Author details

  1. Kerstin Hasenpusch-Theil

    1. Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom
    2. Simons Initiative for the Developing Brain, University of Edinburgh, Edinburgh, United Kingdom
    Contribution
    Data curation, Formal analysis, Supervision, Validation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  2. Christine Laclef

    Sorbonne Université, CNRS UMR7622, INSERM U1156, Institut de Biologie Paris Seine (IBPS) - Developmental Biology Unit, Paris, France
    Present address
    Sorbonne Université, Institut du Fer à Moulin INSERM U1270, Paris, France
    Contribution
    Data curation, Formal analysis, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  3. Matt Colligan

    Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom
    Contribution
    Formal analysis, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6553-8915
  4. Eamon Fitzgerald

    Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom
    Contribution
    Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared
  5. Katherine Howe

    Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  6. Emily Carroll

    Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  7. Shaun R Abrams

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  8. Jeremy F Reiter

    1. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    2. Chan Zuckerberg Biohub, San Francisco, United States
    Contribution
    Conceptualization, Supervision, Funding acquisition, Writing - review and editing
    Competing interests
    No competing interests declared
  9. Sylvie Schneider-Maunoury

    Sorbonne Université, CNRS UMR7622, INSERM U1156, Institut de Biologie Paris Seine (IBPS) - Developmental Biology Unit, Paris, France
    Contribution
    Conceptualization, Supervision, Funding acquisition, Writing - review and editing
    Competing interests
    No competing interests declared
  10. Thomas Theil

    1. Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, United Kingdom
    2. Simons Initiative for the Developing Brain, University of Edinburgh, Edinburgh, United Kingdom
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    thomas.theil@ed.ac.uk
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6590-8309

Funding

Biotechnology and Biological Sciences Research Council (BB/P00122X/1)

  • Thomas Theil

NIH Clinical Center (R01GM095941)

  • Jeremy F Reiter

The Simons Initiative for the Developing Brain (SFARI 529085)

  • Thomas Theil

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are grateful to Drs Thomas Becker, Christos Gkogkas, John Mason, Pleasantine Mill, and David Price for critical comments on the manuscript, and Stéphane Schurmans for the Inpp5eΔ/+ mouse line. We also thank Dr Michaël Trichet (electron microscopy platform of the IBPS-Sorbonne Universités Paris 6) and Dr Alain Schmitt (electron microscopy platform of the Institut Cochin CNRS-UMR 8104) for their help with scanning and transmission electron microscopy analyses, respectively. This work was supported by a grants from the Biotechnology and Biological Sciences Research Council (BB/P00122X/1) and from the Simons Initiative for the Developing Brain (SFARI −529085) to TT and from NIH R01GM095941 to JFR.

Ethics

Animal experimentation: All experimental work was carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and UK Home Office guidelines under the project license numer P53864D41. 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.

Senior Editor

  1. Marianne E Bronner, California Institute of Technology, United States

Reviewing Editor

  1. Fadel Tissir, Universite' Catholique de Louvain, Belgium

Reviewer

  1. Fadel Tissir, Universite' Catholique de Louvain, Belgium

Publication history

  1. Received: April 22, 2020
  2. Accepted: August 24, 2020
  3. Accepted Manuscript published: August 25, 2020 (version 1)
  4. Version of Record published: September 9, 2020 (version 2)

Copyright

© 2020, Hasenpusch-Theil et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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