Cdh2 restricts Notch and Gli1-mediated Shh signaling to ensure proper neocortical development

1. Introduction The neocortex is the most recent acquisition of the mammalian brain (Geschwind & Rakic, 2013) and has experienced great expansion throughout mammalian evolution (Borrell & Reillo, 2012; Molnár et al., 2019; Sousa et al., 2017). It is involved in a wide range of functions, ranging from the integration of the sensory perception, the elaboration and control of voluntary motor programs, language, learning, reasoning or complex thought, among many others (Franco & Müller, 2013). For that, it displays a particular and highly organized structure consisting in the laminar apposition of six cellular layers intermingled by radial or vertical columns (Levitt & Rakic, 1980; P. Rakic, 1972, 2007). These layers harbour an extraordinary neural cell diversity found in many different mammalian species, including mouse and human, in terms of morphology, transcriptomic signature and electrophysiological features, including several subtypes of excitatory projection neurons (PNs), inhibitory interneurons (INs) and macroglia, constituted by astrocytes and oligodendrocytes, have been identified (Johnson & Walsh, 2017; Lodato & Arlotta, 2014; Marques et al., 2016; Mayer et al., 2018; Yuste et al., 2020). During neocortical development, cortical PNs and macroglia are sequentially generated by neural stem cells lining the walls of the lateral ventricles, named radial glial cells (RGCs), from embryonic day 11 (E11) to E17 (Malatesta et al., 2000; Miyata et al., 2001; Noctor et al., 2001). RGCs express astroglial markers and exhibit a polarized radial morphology consisting in basal process elongating towards the pia and an apical process contacting the ventricular surface (Gadisseux & Evrard, 1985; Magdalena Götz & Huttner, 2005; Kriegstein & Alvarez-Buylla, 2009). These apical feet are strongly bound to each other by specialized unions called adherens junctions (AJs) (Shinji Hirano & Takeichi, 2012). Apart from RGCs found in apical locations of the developing neocortex (termed as apical RGCs, aRGCs), in some species, particularly the ones with enlarged neocortices, outer RGCs (oRGCs) also exist in more basal locations (Hansen et al., 2010) (Fietz et al., 2010) (B. Ostrem et al., 2017; Penisson et al., 2019) contributing to neocortical expansion across mammalian evolution (Kalebic & Huttner, 2020; Molnár et al., 2019; Sousa et al., 2017). Together with intermediate progenitor cells (IPCs), derived from RGCs, neural stem cells first give rise to PNs located in the lower neocortical layers mostly projecting to extracortical regions, and then to PNs occupying the upper layers, that mainly establish contacts with other neocortical PNs (Bella et al., 2024; Franco & Müller, 2013; Greig et al., 2013; Lodato & Arlotta, 2014; Molyneaux et al., 2007; Uzquiano et al., 2018). The regulation of the behaviour of RGCs and neural progenitors in terms of proliferation, cell cycle exit and differentiation is crucial for proper neocorticogenesis and depends on a wide variety of molecular players, comprising cell-cell adhesion molecules (CCAMs) such as cadherins, nectins and their related proteins catenins and afadin (Agustín-Durán et al., 2021). The optimal control of these processes is not a trivial matter; on the contrary, alterations in neocortical development underlie the origin of many neuropsychiatric neurodevelopment disorders (NDDs) in humans, that include autism-spectrum disorders (ASD), mental retardation, intellectual disability, bipolarity, schizophrenia or attention deficit and hyperactivity disorder (ADHD) (Akula et al., 2023; Fernández et al., 2016; Juric-Sekhar & Hevner, 2019; Ossola & Kalebic, 2022; Ross & Walsh, 2001). 2. Hypothesis and objectives Among CCAMs, cadherin-2 (Cdh2) and afadin are critical for neocortical development as their absence since early developmental stages of neocorticogenesis triggers RGCs delamination, overproliferation and the generation of a disorganized and enlarged neocortex populated by an increased number of PNs expressing molecular markers typical from the upper neocortical layers (Gil-Sanz et al., 2014). Nevertheless, it has been also reported that the lack of other proteins present at AJs between RGCs, such as β-catenin or Pals1, does not trigger overproliferation nor a subsequent increment of cortical size despite also disrupting AJs (Junghans et al., 2005; S. Kim et al., 2010; Machon et al., 2003; Woodhead et al., 2006). Together with the fact that both a proteolytically-cleaved form of Cdh2 and Afadin can in fact enter the cellular nucleus (Buchert et al., 2007; Luo et al., 2012; Shoval et al., 2006; VanLeeuwen et al., 2014), these evidence suggests that Cdh2 may modulate neocortical development through the regulation of the expression of genes involved in signalling pathways instrumental for the control of RGCs behaviour that ensure adequate neocortical development, preventing the apparition of neurodevelopmental disorders such as ASD. To test our hypothesis, we proposed the following general objectives: I. To identify signaling pathways altered upon Cdh2 deletion in the mouse dorsal telencephalon since early developmental ages. II. To understand the rationale beyond molecular mechanisms governed by Cdh2 in the context of the regulation of RGCs behaviour in terms of cell proliferation, differentiation and cell fate choice. III. To unveil the behavioural consequences of the lack of Cdh2 and its related protein Afadin in the mouse dorsal telencephalon since early developmental ages. 3. Materials and methods For testing our hypothesis through the above-mentioned objectives, we performed a wide array of techniques in wildtype and Cdh2 and afadin conditional knockout (cKO) embryonic and adult mice (Gorski et al., 2002). These include transcriptomic assays such as RNA sequencing (RNAseq) and real-time quantitative PCR (RT-qPCR), molecular cloning, fluorescent in situ hybridization (FISH), immunohistochemistry and immunocytochemistry, in-utero electroporation (IUE) (Saito & Nakatsuji, 2001; H. Tabata & Nakajima, 2001; Hidenori Tabata & Nakajima, 2008), in various vivo pharmacological treatments, primary in vitro cell culture, confocal microscopy and behavioural assessment. 4. Results Objective 1. Identification of signaling mechanisms upon early Cdh2 deletion in the developing neocortex Our RNAseq and RT-qPCR experiments revealed that upon Cdh2 loss in the developing neocortex, Notch and Sonic Hedgehog (Shh) signaling pathways are upregulated at E13.5 in comparison to control embryos. Notch signaling pathway has been widely reporter as key in the control of RGC behaviour during nervous system development in general and cortical development in particular (Corbin et al., 2008; Gaiano et al., 2000; Louvi & Artavanis-Tsakonas, 2006; Mizutani & Saito, 2005; Nian & Hou, 2022). Shh is also involved in the regulation of cortical progenitor behaviour (Cai et al., 2023; Komada, Saitsu, Kinboshi, et al., 2008; Komada, 2012; Palma & Altaba, 2003; Stecca & Altaba, 2005). Besides, its importance in cortical expansion throughout mammalian evolution through the maintenance of cell proliferation and the generation of oRGCs and upper-layer projection neurons (Hou et al., 2021; Komada, Saitsu, Kinboshi, et al., 2008; Komada, 2012; L. Wang et al., 2016; O. R. Yabut & Pleasure, 2018) has been revealed in the last two decades. Interestingly, our data did not show a general upregulation of the expression of Shh genes, but specific increased expression of Gli1 transcription factor and their target genes Fgf15 and Ptch2. Objective 2. Comprehension of the functional relevance of Cdh2-regulated molecular mechanisms for governing RGCs behaviour To evaluate the possible implication of upregulated Notch signaling upon Cdh2 loss in the aberrant behaviour of RGCs and neural progenitors, we performed and an in vitro strategy using DAPT (((2S)-N-[(3,5-Difluorophenyl) acetyl]-L-alanyl-2-phenyl] glycine 1,1-dimethylethyl ester) γ-secretase inhibitor and an in vivo strategy through IUE using a dominant-negative form of RBPJ transcription factor, necessary for the transduction of Notch signaling (Tran et al., 2023). Together, our results showed that Notch antagonism partially rescued the overproliferation trigged by Cdh2 loss during neocortical development. Likewise, we conducted in vivo inhibition of Shh signaling using cyclopamine (J. K. Chen et al., 2002; Heretsch et al., 2010; Lien et al., 2006) in Cdh2 cKO embryos and found that it also rescued cortical overexpansion and overproliferation occurring when Cdh2 is absent during neocortical development. To further understand the importance of upregulated Gli1 expression in this context, we performed Gli1 overexpression experiments by IUE and found that it increases the abundance of proliferative basal progenitors in the developing neocortex, very scarce in the normal mouse embryonic neocortex (Vaid et al., 2018), that seemed to increase the latter generation of upper-layer PNs. Strikingly, we detected a concomitant downregulation of Cdh2 protein expression upon Gli1 overexpression. This led us to conduct additional IUE experiments simultaneously overexpressing Gli1 and Cdh2, and we observed that equivalent increase of Cdh2 and Gli1 expression levels restored normal cortical development. Objective 3. Behavioural consequences of the cortical aberrations due to the lack of Cdh2 and its related protein afadin during neocorticogenesis At the light of and besides our previous results, distinct reasons sustain our interest in examining the extent Cdh2 and its related protein afadin are further critical for normal corticogenesis. First, corticogenesis alterations often trigger neurodevelopmental disorders, such as autism spectrum disorders (ASD), schizophrenia, mental retardation or bipolarity, among many others (Garcia-Forn et al., 2020; Juric-Sekhar & Hevner, 2019; Pinson et al., 2019). Second, some features of Cdh2 and Afadin cortical mutants, including higher cortical size especially appreciable in rostral areas, have been detected in some ASD patients (E Courchesne et al., 2001, 2011; Eric Courchesne et al., 2003, 2011; Redcay & Courchesne, 2005). Third, aberrant numbers of upper-layer-like projection neurons, found in Cdh2 and Afadin mutant cortices, have been associated in mouse with an impaired ability to establish social interactions with other individuals (Fang et al., 2014). And fourth, some polymorphisms found in the human Cdh2 gene are recently starting to be associated with Attention-Deficit/Hyperactivity Disorder (ADHD), obsessive-compulsive and Tourette disorders and even ASD (László & Lele, 2022). Because of these, we conducted behavioural assessment of Cdh2 and afadin cKO pups at P6 and P10, respectively, and found that they emitted lower number of ultrasonic vocalizations (USVs) when separated from their mother and littermates for 5 minutes. As Cdh2 cKO mice do not reach adulthood given the severity of their cortical affectations (Gil-Sanz et al., 2014), we performed additional tests only in Afadin cKO mice. Thanks to our thorough analyses, we detected that these mice exhibited impaired sociability in the social tube test, social interaction test and social three-chamber test. Those were not accompanied by alterations in their motor behaviour, olfaction abilities, their cognitive skills or their non-social anxiety levels. Finally, in order to try to identify the anatomical substrate of these deficiencies and given the involvement in social behaviours of the medial prefrontal cortex (mPFC) (Eric Courchesne et al., 2011; Schubert et al., 2015), we conducted social behavioural assessment in mice with in-mosaic deletion of afadin by IUE. This enabled us to identify similar alterations in sociability, indicating that the mPFC could be an important contributor to the observed deficits in Afadin cKO mice. 5. Conclusions 1) Among the great number of differentially expressed genes identified, Notch- and Shh-related genes are upregulated upon Cdh2 cKO. 2) Notch and Shh downregulation in Cdh2 cKO embryonic neocortex partially rescues cortical overproliferation. 3) Early Gli1 overexpression in the developing neocortex triggers overproliferation and basal progenitor generation, potentially leading to an enhanced generation of ULNs, and downregulating Cdh2 expression. 4) Cdh2 and Gli1 display non-overlapping expression patterns in the whole developing embryo and early Cdh2 overexpression rescues Gli1-overexpression phenotype, restoring normal cortical development. 5) Cdh2 and Afadin cKO mice display similar ASD-like traits in their early days of life consisting in deficient social communication. 6) Adult Afadin cKO mice display other ASD-like traits like social interaction impairments but do not present non-social memory alterations. Such social impairments do not seem to be related with olfaction or enhanced anxiety. 7) Cdh2 and Afadin regulate neocortical development in mice not only by their crucial function as cell adhesion mediators, but also acting as signaling hubs whose absence critically affects how neural progenitors behave, which has an impact on individuals’ behaviour.

Ministerio de Ciencia, Innovación y Universidades

Agencia Estatal de Investigación

Generalitat Valenciana