# SELENOPROTEIN T deficiency alters projection neuron migration during corticogenesis in mice

Emmanuelle Carpentier, Anthony Falluel-Morel, Lisa Brunet, Magalie Bénard, David Alexandre, David Godefroy, Ben Yamine Mallouki, Loubna Boukhzar, Arnaud Arabo, Youssef Anouar

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Emmanuelle Carpentier, Anthony Falluel-Morel, Lisa Brunet, Magalie Bénard, David Alexandre, et al.. SELENOPROTEIN T deficiency alters projection neuron migration during corticogenesis in mice. Neuroscience, 2025, 585, pp.323-334. 10.1016/j.neuroscience.2025.09.015. hal-05326228

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Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
Research Article

SELENOPROTEIN T deficiency alters projection neuron migration during
corticogenesis in mice

Emmanuelle Carpentier<sup>a</sup>, Anthony Falluel-Morel<sup>a,1,◎</sup>, Lisa Brunet<sup>a</sup>, Magalie Bénard<sup>b</sup>, David Alexandre<sup>a,◎</sup>, David Godefroy<sup>a</sup>, Ben Yamine Mallouki<sup>a</sup>, Loubna Boukhzar<sup>a</sup>, Arnaud Arabo<sup>b</sup>, Youssef Anouar<sup>a,∗</sup> ◎

ARTICLE INFO

Keywords:

Corticogenesis

Neuronal migration

Selenoprotein

Antioxidant

A B S T R A C T

During corticogenesis, projection neurons migrate along the radial glial axis to form cortical layers, the alteration of which is associated with functional deficits in adulthood. As byproducts of cell metabolism, reactive oxygen species act as second messengers to contribute to neurodevelopment; however, free radical excess may impede this process. Selenoprotein T (SELENOT) is a newly identified thioredoxin-like enzyme of the endoplasmic reticulum abundantly expressed during embryogenesis whose gene disruption in the brain leads to neuroblast cell demise and neuromorphological alterations due to increased free radical levels. To determine the potential contribution of SELENOT to the establishment of cortical networks, we first analyzed its expression profile in the neocortex at different stages of development using RNA scope *in situ* hybridization. These studies revealed the expression of SELENOT in different cortical layers, and its localization in glutamatergic and GABAergic neurons. Targeted SELENOT gene knockout in the cortex using *in utero* electroporation-mediated gene disruption or Nes-Cre/loxP transgenesis system resulted in an alteration of neuroblast migration polarity, at the level of radial scaffolding, and projection neuron positioning. These results indicate that SELENOT which is highly expressed in the cortex during neurodevelopment plays a crucial role in corticogenesis by promoting projection neuron migration.

Introduction

In mammals, the development of the cerebral neocortex is a partic-
ular histological process that leads to a laminar six-layered cytoarchi-
tecture. This organization is of a main importance for intracortical and
subcortical connectivity in order to ensure the cognitive functions
involving this cerebral structure. During corticogenesis between em-
bryonic day (E) 11 and E19 in mice, immature neurons (neuroblasts) are
born in the ventricular and subventricular zones (VZ and SVZ) by
asymmetric division of radial glial cells (Angevine and Sidman, 1961;
Rakic, 1982; Takahashi et al., 1995; Noctor et al., 2001; Noctor et al.,
2004). After proliferation, neuroblasts migrate along the radial fibres of

radial glial cells from the VZ to the cortical plate (CP) in an “inside-out”
manner. Thus, newly generated neuroblasts terminate their migration
above oldest neuroblasts to form a new cortical layer (Rakic, 1972;
O’Rourke et al., 1992; Nadarajah et al., 2001). The major cell types
found in the cerebral cortex are glutamatergic excitatory neurons and
GABAergic inhibitory interneurons, that account for approximately 80
% and 20 % of total neurons, respectively, and this ratio is maintained
from development to adulthood (Hendry et al., 1987; Sahara et al.,
2012). In contrast to glutamatergic projection neurons which arise from
radial glial cells, GABAergic neurons arise from the ganglionic eminence
of the telencephalon and migrate tangentially to reach the cortex (Tan
et al., 1998; Anderson et al., 1999; Marin et al., 2001). Glutamatergic

Abbreviations: CP, cortical plate; En, embryonic day n; GE, ganglionic eminence; IUE, *in utero* electroporation; ΙΖ, intermediate zone; PND, postnatal day; ROS, reactive oxygen species; SELENOT, selenoprotein T; SP, subplate; SVZ, subventricular zone; VZ, ventricular zone.

* Corresponding author at: U1239, Neuroendocrine, Endocrine and Germinal Differentiation and Communication Laboratory, Rouen-Normandie University, 76821 Mont-Saint-Aignan Cedex, France.

E-mail address: youssef.anouar@univ-rouen.fr (Y. Anouar).

¹ Present address: University of Rouen Normandie, INSERM U1245, Brain and Cancer Genomics, IRIB, F-76000 Rouen, France.

<footnote><sup>a</sup> University of Rouen Normandie, INSERM U1239, Neuroendocrine, Endocrine and Germinal Differentiation and Communication Laboratory, IRIB, CURIB, 76821 Mont-Saint-Aignan, France</footnote>

<footnote><sup>b</sup> HeRacLeS, Inserm US 51, CNRS UAR 2026, IRIB, CURIB, 76821 Mont-Saint-Aignan, France</footnote>
neuron population is responsible for information treatment and storage through their specific connectivities, depending on the cortical layer where they reside (Kwan et al., 2012), and the activity of these neurons is regulated by GABAergic neurons. Alteration of cortical neuroblast migration contributes to neurodevelopmental deficits such as autism spectrum disorder (ASD) or schizophrenia (Wegiel et al., 2010; Stachowiak et al., 2017).

Extrinsic and intrinsic factors are involved in proper cell proliferation, migration and differentiation to allow the development of the neocortex. Among these, reactive oxygen species (ROS) which are by-products of cell metabolism can also act as second messengers to stimulate, for instance, self-renewal of neural stem cells, in specific time-windows and localizations (Le Belle et al., 2011; Chui et al., 2020). However, uncontrolled ROS excess leads to oxidative stress, an aggravating factor of pathologies, which can provoke the loss of the axon initial segment, a decrease in adhesion molecule expression or cell death (Fatokun et al., 2008; Arai et al., 1998; Clark et al., 2017).

In order to protect the brain against ROS and to maintain redox homeostasis, numerous antioxidant systems, including key enzymatic activities carried by selenoproteins, are recruited by nerve cells. Selenoproteins are a particular class of enzymes that contain selenium in the organic form of the amino acid selenocysteine (Sec). Selenoproteins are classified in different families, such as glutathione peroxidases (GPX), thioredoxin reductases (TXNRD), and iodothyronine deiodinases (DIO), according to their antioxidant activities mediated by the Sec residue present in their catalytic site. Sec-containing enzymes are crucial for development since their global disruption is lethal in mice (Bösl et al., 1997). Moreover, previous findings showed that selenoproteins are abundantly expressed during embryogenesis and that, in the brain, cortical and hippocampal neurons are the favourite site of their expression (Jakupoglu et al., 2005; Zhang et al., 2008). It is now admitted that absence or reduction of selenoprotein expression could contribute to severe neurodevelopmental disorders and neurodegenerative diseases such as ASD, seizure or Parkinson disease (Wirth et al., 2010; Raymond et al., 2014; Cardoso et al., 2015).

The precise functions of certain of these enzymes are not fully elucidated. This is the case of selenoprotein T (SELENOT), a selenoprotein highly conserved during evolution (Moustafa and Antar, 2012), which exerts oxidoreductase activity and whose gene disruption is lethal early during embryonic development in mice (Boukhzar et al., 2016). SELENOT is required for calcium mobilization (Grumolato et al., 2008; Mazza et al., 2019) and plays a role in protein N-glycosylation in neuroendocrine cells (Hamieh et al., 2017). In the brain, SELENOT has protective effects during development and after injury by alleviating oxidative stress (Boukhzar et al., 2016; Castex et al., 2016; Alsharif et al., 2021). SELENOT is localized in immature and differentiating neurons of the rat neocortex (Tanguy et al., 2011), and its conditional gene knockout in the mouse brain causes neuroblast cell death and several neuroanatomical alterations (Castex et al., 2016). In fact, mice with a brain-specific SELENOT knockout displayed a hyperactive phenotype and a higher vulnerability to neurotoxic agents (Boukhzar et al., 2016). Together, these observations strongly suggest that this selenoenzyme plays an important role during neurodevelopment, which may include corticogenesis.

To elucidate the role of SELENOT in neurodevelopmental processes such as corticogenesis, we sought first to establish its expression pattern in the neocortex to potentially link the expression profile to the development of subcortical structures. Then, we genetically disrupted SELENOT expression in radial glial cells and neuroblasts of developing transgenic mice carrying a SELENOT-floxed gene to evaluate the role of this selenoprotein during corticogenesis. The data obtained showed that SELENOT is widely expressed at all layers of the neocortex at different stages of corticogenesis and that its deficiency alters neuroblast migration and projection neuron positioning.

# Materials and methods

## Animals

All animal experiments were approved by the Normandy Ethics Committee on Animal Experimentation (Authorization No. 25444, No. 6637 and No. 16178) and were performed in accordance to the European Committee Council Directives. C57BL/6J mice were obtained from Janvier Labs (Saint Berthevin, France). SELENOT-floxed (SELENOT<sup>f/f</sup>) mice were obtained by internal colony breeding of genitors with a SELENOT<sup>f</sup> allele produced in collaboration with the Mouse Clinical Institute (Strasbourg, France). Transgenic SELENOT<sup>f</sup> mice were generated by homologous recombination of C57BL/6J genome sequence encoding the active site of the protein, between exons 2 and 3. Nes-Cre/SELENOT<sup>f/f</sup> KO mice were obtained by breeding of SELENOT<sup>f/f</sup> mice with Tg (Nestin-Cre)1 Kln +/- knock-in mice, acquired from the Jackson Laboratory (Bar Harbord, ME, USA) and lack of SELENOT in brain of KO mice was previously confirmed (Boukhzar et al., 2016; Castex et al., 2016). Mice were housed under controlled conditions with a 12-h light/12-h dark cycle (light on at 07:00 am). Animals had access to food and water *ad libitum* and were maintained at 22 ± 2 °C ambient temperature. Embryos were electroporated and/or surgically removed from SELENOT<sup>f/f</sup> pregnant females, and embryonic stages were determined by the day of appearance of the vaginal plug, as E0.5. Genotypes were obtained by PCR of genomic DNA from tail clips with the following primers: forward, 5'-GGCTTTATGTAAGCAGTTCTAAACTGTTTCTGC-3'; and reverse, 5'-CGCCCCATTTTATAAACTTTGTATGTTTATGCCC-3', for WT (211 bp) and SELENOT<sup>f</sup> (261 bp) alleles; forward, 5'-GGCTTTATGTAAGCAGTTCTAAACTGTTTCTGC-3'; and reverse, 5'-GCCTAGGTTT-TACCTGAGAA ACCAAAGG-3', for the SELENOT allele (409 bp).

## RNAscope® in situ hybridization

Mice were anesthetized using a solution of ketamine (100 mg/kg) and xylazine (10 mg/kg), and perfused with sterile phosphate-buffered saline (PBS) followed by sterile paraformaldehyde (PFA) 4 %. Brains were dissected and post-fixed in PFA 4 % overnight at 4 °C. A cryoprotection was performed through successive immersion in sterile 15 % and 30 % sucrose in PBS. Brains were frozen in isopentane in dry ice and stored at -80 °C. Tissues were sectioned into 10–12-µm slices with a cryostat. Slices were washed 5 min in sterile PBS and heated at 60 °C in a dedicated oven (ACD HybEZ™ II Hybridization System (220v) with ACD EZ-Batch Slide System, Biotechne, Rennes, France) under Bake mode during 30 min, and then post-fixed during 15 min with sterile PFA 4 %. Slices were dehydrated in graded solutions of ethanol at 50 %, 70 % and twice 100 % during 5 min for each bath. Slices were covered with H₂O₂ during 5 min and washed with distilled H₂O two times. Slices were submerged 10 sec in distilled water at 99 °C and 5 min in RNAscope 1X Target Retrieval Reagent (ACD Biotechne) at 99 °C. Slides were rinsed 15 sec in distilled water at RT, submerged 3 min in ethanol 100 % and dried at RT. Slices were covered with protease RNAscope (ACD Biotechne) during 30 min at 40 °C and washed with distilled water two times. Hybridization was performed with 50 µl/slice of probe mix during 2 h at 40 °C in an oven. Probes used were supplied by ACD Biotechne and were as follows: SELENOT probe targeting the region 1428–3520 (NM_001040396.3), designed in C2; glutamate transporter targeting VGLUT2 (Slc17a6), designed in C3; GABA transporter targeting VGAT (Slc32a1), designed in C3. After hybridization, slices were covered with RNAscope Multiplex FL v2 Amp 1 during 30 min at 40 °C and washed with RNAscope wash buffer two times at RT, then covered with RNAscope Multiplex FL v2 Amp 2 during 30 min at 40 °C and washed with RNAscope wash buffer two times at RT, and finally with RNAscope Multiplex FL v2 Amp 3 during 15 min at 40 °C and washed with RNAscope wash buffer twice at RT (RNAscope Multiplex FL v2 Amp 1,2,3: RNAscope® Multiplex Fluorescent Reagent Kit v2; ACD Biotechne). After amplification, the staining steps were carried out sequentially.
Slices were covered with RNAscope Multiplex FL v2 HRP-C1 (ACD Biotechne) during 15 min at 40 °C and washed at RT with RNAscope wash buffer twice, then covered by 80 µL of Opal 520 at 1/1500 (Opal 520 reagent pack, FP1487001KT, AKOYA Biosciences, Malborough, United States) during 30 min at 40 °C and washed at RT with RNAscope wash buffer twice, followed by staining of C2 to block with HRP blocker (ACD Biotecne) during 15 min at 40 °C and washed at RT with RNAscope wash buffer twice. Slices were covered by 80 µL of Opal 650 at 1/1000 (Opal 650 reagent pack, AKOYA Biosciences) during 30 min at 40 °C and washed at RT with RNAscope wash buffer twice, and staining of C3 to block with HRP blocker (ACD Biotecne) during 15 min at 40 °C and wash at RT with RNAscope wash buffer twice. Doublecortin was then labeled by immunostaining, as described in the immunohistofluorescence methods. Staining of the nuclei was done with RNAscope® 4-6-diamino-2-phenylindole (DAPI, ACD Biotechne), and the slides were mounted with fluoromount (Sigma-Aldrich, Saint-Quantin Fallavier, France). Images were acquired using the Leica Thunder 3D DCV microscope (Leica microsystems, Nanterre, France) and were processed under ImageJ/Fiji software.

## *In utero electroporation*

*In utero* electroporation was performed in homozygous female mice carrying a floxed SELENOT gene (SELENOT<sup>fl/fl</sup>), at 14.5 gestational day which corresponds to the peak of neurogenesis (Takahashi et al., 1995). Females were anaesthetized through isoflurane inhalation during surgery. To prevent peri-/post-surgery pain, buprenorphine at 0.1 mg/kg was injected subcutaneously 10 min before surgery and uterine horn exposition. One µg of pCAG-IRES-eGFP plasmid DNA (control plasmid (Hand et al., 2005; Megason and McMahon, 2002), or pCAG-CRE-IRES-eGFP (abbreviated as pCAG-CRE) where the Cre enzyme was inserted after excision from pSK-Cre1 plasmid (provided by Clifford Tabbin, Harvard Medical school), was diluted in PBS containing 0.05 % of Fast Green (Sigma-Aldrich) and injected into the lateral ventricle of embryos using a Hamilton syringe with a 33 Gauge needle (Dutsher, Issy-les-Moulineaux, France). Each embryo was placed between tweezer-type electrodes (CUY 650P5, Sonidel, Dublin, Ireland) linked to an electroporator (CUY21 EDIT, Sonidel, Dublin, Ireland) to apply 5 pulses at 50 V/50 ms, separated by 950 ms intervals. Uterine horns were placed back into the mice abdomen and the cavities were sutured-closed. Our electroporation success rate was about 40-50 %. Unsuccessful cases were related to embryonic death, absence of eGFP expression in neuroblasts or misplaced expression of eGFP probably due to inadequate electrode positioning. The sex of the embryos was ultimately determined by analyzing SRY gene expression by PCR, and we have not observed any significant difference between males and females in terms of SELENOT role in corticogenesis.

## *Immunohistofluorescence*

Embryo heads were collected 24 or 72 h after *in utero* electroporation and fixed in 4 % PFA, for 6 h at 4 °C. Samples were then dehydrated by immersion in 15 % sucrose/PBS overnight at 4 °C, and in 30 % sucrose/PBS overnight at 4 °C before being embedded in tissue-freezing medium (Tissue-Tek, Sigma-Aldrich) and stored at - 80 °C. Immunohistofluorescence was performed on 12-µm coronal sections obtained by cryostat microtome (Leica). For SELENOT, nestin and CTIP2 protein staining, slides were post-fixed in 4 % PFA for 30 min and blocked in PBS containing 1 % bovine serum albumin, 0.3 % Triton X-100, 1 % donkey serum /for 2 h at RT. The antibodies anti-SELENOT (1:200, (Grumolato et al., 2008)), anti-nestin (1:250, Neuromics, Edina, USA), anti-DCX (1:250, Santa Cruz Biotechnology, Germany), anti-CTIP2 (1:100, Invitrogen, Illkirch, France) and anti-Cux-1 (1:200, Fisher Scientific, Illkirch, France) were incubated overnight at 4 °C. Visualization of immunostaining was performed by Alexa Fluor 650 or 594-conjugated secondary antibodies diluted at 1:500 (Invitrogen). Nuclei were

visualized with DAPI (Sigma-Aldrich) at 1 mg/ml in PBS, and slides were mounted with PBS/glycerol 50/50 v/v for observation. Images were acquired with Leica Thunder 3D DCV microscope (Leica microsystems) and were processed using ImageJ/Fiji software.

## *Statistical analysis*

Data comparisons were performed using the results of at least three independent experiments and were represented as means ± SEM. Statistical analysis was performed on Graphpad Prism 5 software, Mann-Whitney *U* test or Unpaired *t* test. Significance was set at \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 and \*\*\*\*p < 0.0001.

## *Results*

### *SELENOT is expressed in proliferating and immature layers of the neocortex within glutamatergic and GABAergic-positive neurons*

To define SELENOT expression profile at the histological level in the neocortex, we used RNAscope *in situ* hybridization analysis. Fig. 1 shows representative SELENOT mRNA staining in all neocortical layers at embryonic stages E14.5 and E17.5, including proliferative (VZ and SVZ), migrating (IZ and SP) and differentiating zones (CP). SELENOT mRNA was also expressed in all cortical layers at birth, PND 10, PND 20, PND 60, 6 months and 1 year of age (Fig. 1). The intensity of SELENOT mRNA signal was virtually similar in brain slices of adult and aged mice compared to embryonic and early postnatal stages (Fig. 1). However, no reliable mRNA signal quantification could be performed since the SELENOT mRNA was abundant and diffuse in the tissue sections.

In order to determine the neuronal cell types expressing SELENOT gene in the different embryonic cortical layers and to be able to link these data to the following functional studies, we analyzed its co-expression in embryos (E17.5) with γ-aminobutyric acid (GABA)-ergic and glutamatergic neurons by *in situ* hybridization or with DCX by *in situ* hybridization/immunohistochemistry (Fig. 2). Co-localization studies showed that SELENOT is present in GABA-positive neurons, labeled with vesicular GABA transporter (VGAT) probes, in the ganglionic eminence where GABAergic neurons are generated, and in the different embryonic cortical layers where GABAergic neuroblasts migrate during corticogenesis (Polleux et al., 2002) (Fig. 2a, b, d). Within cortical layers, SELENOT mRNA signal was present in the VZ where radial glial cells are present to generate neuroblasts (Molyneaux et al., 2007), but also in differentiating neurons labeled with an antibody against doublecortin (DCX), in the CP (Fig. 2a, c, d) (Molyneaux et al., 2007). In addition, SELENOT was present in glutamatergic-positive neurons, stained with type-2 vesicular glutamate transporter (VGLUT2) probes, in all cortical layers (i.e. proliferative, migration and differentiation layers) (Fig. 2e, f, g).

### *Impairment of SELENOT expression in neuronal progenitors through in utero electroporation (IUE) leads to neuroblast positioning defects*

As SELENOT is expressed during embryogenesis in the neocortex including proliferative and immature layers, we sought to determine its contribution to corticogenesis and more particularly in neuroblast migration. To this intent, we first investigated whether SELENOT deficiency could lead to neuronal loss during embryogenesis, by counting the number of Caspase-3-positive cells in cortical areas at E14.5. This analysis revealed that SELENOT gene disruption had no significant effect on cortical neurogenesis at this developmental stage (Fig. S1). We then investigated the impact of SELENOT deficiency on cortical neuroblast migration. For this, IUE was carried out in SELENOT<sup>fl/fl</sup> mice to deliver a plasmid containing a Cre recombinase into the germinative neuroepithelium bordering the lateral ventricle of embryos at E14.5, when neuroblasts are actively generated (Takahashi et al., 1995; Smart and McSherry, 1982). We electroporated the Cre-encoding plasmid in
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Analysis of SELENOT mRNA expression in cortical layers throughout mouse development using RNAscope *in situ* hybridization. Representative images showing SELENOT mRNA from E14.5 to 1-year are presented. Staining with DAPI (blue) was used to evaluate cell density. SELENOT (grey) is expressed at similar levels in all cortical layers and between embryonic, PND, adult and aged mice. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; GE, ganglionic eminence; LV, lateral ventricle; WM, white matter. Scale bar 150 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

the border of the lateral ventricle in order to disrupt SELENOT gene in neuronal progenitors (Fig. 3a). Both control and pCAG-CRE plasmids carried a gene encoding an enhanced green fluorescent protein (eGFP) to visualize transfected cells (Fig. 3b). Fig. 3c shows the efficiency of the electroporation with green fluorescent cells visible in cortical layers, for the control and pCAG-CRE plasmids. The efficiency of SELENOT gene disruption was assessed by PCR on cortical tissue of embryonic mice after IUE, and this analysis showed the presence of the SELENOT<sup>fl/fl</sup> allele only in the neocortex transfected with the pCAG-CRE plasmid (Fig. S2), thus showing the disruption of the SELENOT gene in cells present in this tissue. We also performed an immunostaining of SELENOT which revealed the significant reduction of SELENOT in cortical layers after pCAG-CRE transfection compared to control (Fig. 3c). Thus, in control condition, SELENOT was detected in a high proportion of fluorescent cells as it could be expected (Fig. 3d, e). IUE with the pCAG-CRE plasmid led to a 45% decrease (t = 5.40, df = 4, **p = 0.0057); n = 522 cells for control and n = 935 cells for pCAG-CRE condition) in the number of SELENOT-positive fluorescent cells in the neocortex compared to animals who received the control plasmid (Fig. 3e), thus confirming the SELENOT knockout, albeit partial, in Cre-expressing neuroblasts.

Seventy-two hours after IUE (E17.5), most fluorescent cells transfected with pCAG-CRE were observed in the IZ layer, while most fluorescent cells transfected with the control plasmid were located in the CP (Fig. 4a). Quantification of eGFP-positive cells showed a 148% increase

(t-value = 4.28, df = 8.43, **p = 0.0024, n = 6–7) in the proportion of cells located in the IZ following SELENOT disruption and, conversely, a 37% decrease (t-value = 4.14, df = 8.27, **p = 0.0031, n = 6–7) in the percentage of fluorescent cells in the CP (Fig. 4b). Of note, a small proportion of neuroblasts transfected with pCAG-CRE could reach the CP, probably due to compensatory mechanisms in these cells.

*Migrating neuroblast polarity is altered in the absence of SELENOT*

To better understand the neuroblast positioning defect observed after SELENOT disruption, i.e. accumulation of deficient cells in the IZ, we analyzed the orientation of their leading process which normally drives their migration through the radial glia axis up to the neocortex (Nadarajah and Parnavelas, 2002) (Fig. 5a,b). Measuring the angle (degrees) between the neuroblast leading process and the main axis of radial glial cells (Fig. 5b) and representing the cumulative distribution of these angles in each cortical layer, we found that most SELENOT-deficient neuroblasts display a higher angle of migration in the VZ/ SVZ (t = 2.50, df = 27.92, *p = 0.0186; n = 12–18 cells per condition) and IZ (t = 11.15, df = 388, ****p < 0.0001, n = 273–267 cells per condition) compared to cells transfected with the control plasmid (Fig. 5c, d). Neuroblasts that successfully reach the CP have no migration angle alterations (t = 1.07, df = 196, p = 0.2861, Fig. 5e).
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Expression of SELENOT in glutamatergic and GABAergic cells during embryogenesis. **a, d** Representative images showing the localization of SELENOT (grey) at E17.5 in the proliferative (VZ/SVZ), the migrating (IZ) and the CP layers of the mouse neocortex, delimited by dashed lines after DAPI staining (blue). **a, b, d** SELENOT was detected in GABAergic neurons (VGAT; magenta) in the GE where these neurons are generated and in all cortical layers, where GABAergic neurons migrate. **a, c, d** In the CP, SELENOT was found in differentiating DCX-positive immature neurons (red). **e, f, g** SELENOT was also detected in glutamatergic neurons (VGlut2, green) in all cortical layers (delimited by DAPI staining, blue) where they proliferate (VZ/SVZ), migrate (IZ) and differentiate (CP). Images in the insets correspond to a higher magnification of the zones delimited by squares in the main photomicrographs. VZ, ventricular zone; SVZ, subventricular zone; IZ, inter-mEDIATE zone; CP, cortical plate; GE, ganglionic eminence; LV, lateral ventricle. Scale bars 150 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

Absence of SELENOT leads to radial axis abnormalities

Since IUE was performed in the border of the lateral ventricle at E14.5, the plasmid is most likely transfected in radial glial progenitors as well which serve as a scaffold for neuroblast migration (Rakic, 1972). We thus hypothesized that alteration of neuroblast migration could be due to disruption of radial glial scaffolding after SELENOT gene knockout. To investigate this possibility, we first confirmed the localization of SELENOT in radial glia cells in both their soma and processes, using an antibody directed against nestin (Fig. 6a). Then, we used nestin labeling of radial glia cells to analyze the morphology of their processes in the cortical layers 24 h after IUE of Cre-expressing or control plasmids. Although the full length of each process could not be determined in the tissue sections analyzed, we could quantify the random apparent length of these processes within each layer of SELENOT-deficient compared to control animals (Fig. 6b). This analysis revealed a decrease of 46 % in this parameter in VZ/SVZ (****p < 0.0001, n = 8–9 processes, Mann-Whitney U test), 20 % in IZ (*p = 0.0164, n = 13 processes, Mann-Whitney U test) and 33 % in CP (**p = 0.0079, n = 8–9 processes, Mann-Whitney U test, Fig. 6c).

SELENOT is required for the proper positioning of deep layer neurons in adult neocortex

As SELENOT deficiency after IUE alters neuroblast polarity as well as radial glial scaffolding, we sought to determine the positioning of

SELENOT-deficient neurons in the neocortex of Nes-Cre/SELENOT<sup>f/f</sup> adult mice, where SELENOT was disrupted in neural cells. To do this, we immunostained the neocortex with an antibody directed against BAF Chromatin Remodeling Complex Subunit (BCL11B or CTIP2), a transcription factor primarily expressed in neurons of the deep layers (V and VI), and preferentially in layer V (Hevner, 2007; Chen et al., 2008). Indeed, CTIP2 immunolabeling was observed in layers V and VI of the adult neocortex in Nes-Cre/SELENOT<sup>+/+</sup> mice, with a stronger labeling in layer V (Fig. 7a). In contrast, CTIP2-positive cells were more abundant in layer VI and less in layer V in adult SELENOT-deficient mice (Fig. 7a). Analysis of CTIP2 distribution in adult Nes-Cre/SELENOT<sup>f/f</sup> neocortex showed a decrease of 5 % and 23 % in the C4 and C5 layers (t = 3.14, df = 3.30, *p = 0.0455; t = 8.27, df = 3.64, **p = 0.0017, n = 3), respectively, and an increase of 17 % in the C6 layer (t = 17.50, df = 2.88, ***p = 0.0005, n = 3), compared to adult Nes-Cre/SELENOT<sup>+/+</sup> mice (Fig. 7b). These data confirm the results obtained with IUE, thus demonstrating that SELENOT plays a physiological role in cortical lamination. We also immunostained the neocortex with an antibody directed against Cux-1 (Cut-Like Homeobox 1), a DNA binding protein preferentially localised in upper cortical layers (Nieto et al., 2004). However, no positioning defects were observed for Cux-1-positive neurons (Fig. 7c), and quantitative results confirmed that these neurons are correctly established at the superficial neocortex for both WT and Nes-Cre/SELENOT<sup>f/f</sup> adult mice (Fig. 7d).
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Efficacy of SELENOT disruption by *in utero* electroporation (IUE). a) IUE was performed in E14.5 embryos whose SELENOT gene was floxed (SELENOT flox/flox). Plasmids were injected in the lateral ventricle of embryos and transfected in neuroblast progenitors located at the border of the ventricle (VZ/SVZ) by electric pulses through tweezer-type electrodes as indicated by +/- signs. Embryos were collected 24 h (E15.5) or 72 h (E17.5) after IUE. b) The control plasmid is a pCAG backbone containing an eGFP sequence to visualize the transfected cells, while the plasmid designed to invalidate the SELENOT gene (pCAG-CRE) contained, in addition, a gene encoding a Cre recombinase which triggers cyclization and recombination of the floxed part of the SELENOT gene in SELENOT flox/flox embryos. c) Representative images of transfected cells (green) in E17.5 embryos showing the expression of SELENOT (grey) in control condition (Control) and the reduction of SELENOT labeling in all cortical layers after Cre expression in pCAG-CRE-electroporated neocortex (pCAG-CRE). Nuclei were labeled with DAPI (blue). Images in (d) show the colocalization of SELENOT within eGFP-positive cells transfected with the control plasmid (arrow) but also its absence in other eGFP-positive cells (arrowhead). Acquisitions were obtained by Z-stack imaging. e) Quantification of the signal revealed a 45 % decrease of SELENOT-positive fluorescent cells when electroporation was performed with the pCAG-CRE plasmid compared to control. Data are represented as mean ± SEM, n = 522 cells for control and 935 cells for pCAG-CRE conditions, \*\*p < 0.01 using Unpaired t test. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; LV, lateral ventricle. Scale bar 10 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

## Discussion

SELENOT is expressed in the cerebral cortex during mouse lifespan

Our previous studies have shown a role of SELENOT in the onto-genesis of the brain in mice (Castex et al., 2016), but the role of this novel selenoenzyme in the different cerebral ontogenetic processes such as corticogenesis remains unknown. Here, we report the importance of SELENOT for normal neuroblast migration during corticogenesis. To understand its contribution for the development of the different cortical layers, we first analyzed SELENOT expression profile in the mouse cerebral cortex, using the RNAscope *in situ* hybridization method. Our data indicate that SELENOT is expressed in the mouse brain early during embryogenesis, confirming our previous results obtained in the rat (Tanguy et al., 2011). In the mouse cerebral cortex, SELENOT mRNA expression is found in all layers from embryogenesis to 1 year-old, and at similar levels at each stage. These gene expression data strongly suggest that SELENOT could play a role in the neocortex during its ontogenesis but also later during brain aging. This observation along with our previous data showing that SELENOT can protect neuroblasts and neurons against oxidative stress (Boukhzar et al., 2016; Castex et al., 2016; Abid et al., 2019; Jehan et al., 2022), suggests that SELENOT expression is

associated with defense mechanisms against ROS-producing processes which occur in the developing but also in the aged brain (Perez-Campo et al., 1998; Passos et al., 2010).

SELENOT is present in proliferative and differentiating neuroblasts

RNAscope *in situ* hybridization analysis showed that SELENOT is present in the ventricular and cortical plate layers. This finding suggests that the selenoprotein could be an important actor in the ventricular zones where cell proliferation occurs during embryogenesis. It has been shown that SELENOT expression is crucial to maintain nerve cell division, by promoting cell cycle G1-S transition (Shao et al., 2019). Moreover, the presence of SELENOT in DCX-labeled immature neurons that reside in the cortical plate at E17.5, may also reflect the importance of this protein for neuroblast differentiation. In accordance, previous studies have shown that SELENOT is overexpressed in differentiating cells and participates in calcium mobilization, a process necessary for cell differentiation and specification (Grumolato et al., 2008; Tonelli et al., 2012). As suggested above, SELENOT could act through its cyto-protective action at different spatio-temporal levels to contribute to neurogenesis.

We found that SELENOT mRNA is present in both glutamatergic and
<figure><img src="image_5.png" /><figcaption>Fig. 4. Disruption of SELENOT expression by IUE leads to neuroblast positioning defect. a) Representative images obtained 72 h after IUE (E17.5) with the control or pCAG-CRE plasmid. In control condition, most eGFP-positive electroporated cells were visible in the CP, while a higher proportion of eGFP-positive cells remained in the IZ when embryos were electroporated with the pCAG-CRE plasmid. Cell nuclei were stained with DAPI (blue). b) Quantification of eGFP-positive neuroblasts in each cortical layer after electroporation of embryos with the two different plasmids. Data are represented as mean ± SEM, n = 6 for control and n = 7 for pCAG-CRE, **p < 0.01 using Unpaired t test. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar 50 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

GABAergic neuroblasts. Glutamatergic cells expressed SELENOT in all cortical layers, comprising proliferative (VZ, SVZ), migrating (IZ) and differentiating (CP) layers, at E17.5. In fact, glutamate is essential for different developmental processes, depending on the cell localization. Thus, glutamate stimulates neuroblast proliferation in the VZ, but not in the SVZ (LoTurco et al., 1995; Haydar et al., 2000; Luk and Sadikot, 2004), and neuroblast migration toward the CP, via a chemoattractant effect mediated by NMDA receptors and increased intracellular calcium (Behar et al., 1999; Simonian and Herbison, 2001). However, another study showed that the stimulation of NMDA receptors in the cortex acts as a stop signal for migration (Kihara et al., 2002). These results indicate that glutamate exerts opposite effects during corticogenesis, depending on the time-window and neuroblast localization. SELENOT may play a role in glutamatergic cells during corticogenesis to allow appropriate production of this essential neurotransmitter, as it was previously shown in dopaminergic neurons (Boukhzar et al., 2016). SELENOT was also detected in cortical GABAergic neurons, which are generated in

ganglionic eminences in the ventral telencephalon and which reach the cortex by tangential migration (Molyneaux et al., 2007). SELENOT is detected in the site of origin of these cells and in generated and migrating GABAergic neuroblasts, at E17.5. These data suggest that SELENOT could play a role in the implementation of GABAergic neurons. Thus, SELENOT expression in neuronal cells in the developing cortical layers could be involved for instance in the protection of different cortical cells against oxidative stress which is inevitably produced during the different processes, i.e. cell proliferation, migration and differentiation, of cortical lamination.

SELENOT is required for neuroblast migration

In agreement with our previous data (Castex et al., 2016), we did not observe any significant effect of SELENOT deficiency on the number of Caspase-3-positive cells in cortical layers at E14.5, suggesting that SELNOT is not essential for neuroblast survival at this stage. However,
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** SELENOT deficiency in neuroblasts leads to migration polarity abnormalities. a) Transfected neuroblasts (green) migrate along the radial glial axis from the VZ to the CP. The inset shows leading processes of neuroblasts (arrows), which drive their migration. b) The migration polarity of neuroblasts was determined by calculating the angle (α in degrees) between the radial glial axis and the leading process of neuroblasts as shown. The graphs represent the cumulative distribution of the angles measured for cells transfected by the control (blue) or pCAG-CRE (red) plasmids. Seventy-two hours after electroporation, pCAG-CRE cells have a significantly higher angle of migration in VZ/SVZ (c) (n = 12–18 cells per condition) and IZ (d), (n = 267–273 cells per condition), compared to control cells. (e) Cells that reach the CP display no difference between the two groups (n = 117–292 cells per condition). Only cells with a distinguishable leading process were included in this analysis. \*p < 0.05; \*\*\*p < 0.0001; ns, not significant versus the respective control using Unpaired t test. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bar 50 µm and zoom 10 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

we have previously reported a decrease in neuroblast cell number at post-natal day 7, which is compensated at adulthood (Castex et al., 2016). These observations indicate that SELENOT could play a role in neuroblast cell survival only at specific developmental windows. To elucidate the possible contribution of SELENOT to neuroblast migration during corticogenesis, we performed *in utero* electroporation of a Cre-expressing plasmid in mice to inactivate SELENOT gene in floxed neuroblast progenitors. We found that significantly less neuroblasts reach the CP 72 h post-transfection, compared to control, and numerous SELENOT-deficient cells remained in the IZ. Nevertheless, not all transfected cells were impacted since a certain number could be observed at the superficial layer. This could be ascribed to the level or timing of Cre recombinase expression in transfected cells, as attested by different eGFP staining levels among the cells. Alternatively, these cells may have sufficient amounts of SELENOT produced before transfection that allow them to cope with the post-transfection SELENOT deficiency.

During corticogenesis, radial glial cells give rise to neuroblasts and extend long processes known as the radial axis, which serve as a scaffolding for neuroblast migration until the CP (Rakic, 1972). Neuroblasts are guided by the radial axis, notably thanks to adhesive molecules (Anton et al., 1999; Sanada et al., 2004). Since SELENOT-deficient neuroblasts exhibited an altered positioning during corticogenesis, we investigated the angle of neuroblast migration with respect to the radial axis. We found that these neuroblasts located in the VZ/SVZ and IZ have a larger angle of migration compared to control. This means that

neuroblasts do not migrate properly towards the CP, and can explain the increase in the number of cells accumulating in the IZ. This abnormality could result from a loss of adhesion between neuroblasts and the radial axis or even from glial scaffolding alterations. In fact, SELENOT is expressed in radial glial cells and its disruption could affect scaffolding. Therefore, we analyzed the morphology of glial processes using nestin as a marker, and found an overall decrease in their apparent length with a disorganised scaffold. These data suggest that neuroblast migration is altered due to a loss of neuroblast polarity, probably as a result of radial glial scaffolding impairment. Interestingly, a role of SELENOT in cell adhesion has been previously described in fibroblasts (Sengupta et al., 2009), and may also occur in neuroblasts and account for their altered positioning and migration after disruption of the selenoprotein gene. Besides, it was previously reported that SELENOT is required for glycosylphosphatidylinositol (GPI)-anchored protein synthesis (Liu et al., 2018). GPI anchoring of proteins is a post-translational modification that occurs in the endoplasmic reticulum (Ferguson et al., 1985) and concerns about 150 cell surface proteins in mammals, including neural adhesive molecules (Karagogoeos, 2003). The role of SELENOT in this process could be related to its implication in protein glycosylation that we previously described (Hamieh et al., 2017), since N-glycan moieties of GPI-anchored proteins play an important role in their transport and their calnexin/calreticulin quality control cycle. It is thus tempting to speculate that the lack of SELENOT leads to N-glycosylation defects in GPI-anchored proteins including adhesive proteins present in radial glial
<figure><img src="image_7.png" /><figcaption>**Fig. 6.** SELENOT disruption in radial glial progenitors leads to alteration of radial glial scaffolding. a) Immunostaining for nestin (red) was performed in control condition to confirm the expression of SELENOT (grey) in radial glial soma and processes. Z-stack imaging shows the colocalization of SELENOT with nestin (white arrows). b) Radial glial scaffolds in neocortical slices of E17.5 mice were compared between eGFP-positive cells transfected with control (upper panel) or pCAG-CRE (lower panel) plasmids. Radial glial processes have a polarized organization toward the pial surface in the normally developing neocortex, but are more disorganised and shorter in pCAG-CRE-treated neocortex (compare images with nestin staining where processes are indicated by vertical dotted lines). Nuclei are labeled with DAPI. c) Quantification of radial glial processes length indicates a decrease in radial glial processes length throughout the different cortical layers after transfection with pCAG-CRE plasmid, with 46% decrease in VZ/SVZ (n = 8–9 processes), 20% decrease in IZ (n = 13 processes) and 33% decrease in CP (n = 8–9 processes). Quantification was performed on Z-stack projection and represents means ± SEM. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.0001 using Mann-Whitney *U* test. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; LV, lateral ventricle. Scale bar 10 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

fibres and in neuroblasts, which in turn could lead to impairment of glial-cell adhesion and loss of polarity for neuroblasts. Thus, the phenotype observed could be due to improper cell surface protein expression disrupting the radial glia/ neuroblast interactions leading to frequent detachment and overall mispositionning of the migrating cells. Alternatively, it is also possible that this imperfect cell orientation may arise from the initial mitotic spindle orientation that normally leads to symmetric/asymmetric cell division.

These results obtained after disruption of SELENOT through IUE highlight its crucial role for the positioning of projection neurons, through neuroblast polarity maintenance and radial glial scaffolding. Interestingly, analysis of the distribution of the profound layer marker CTIP2 showed a mispositioning of deep cortical neurons in Nes-Cre/SELENOT<sup>f/f</sup> adult mice, while the upper cortical neurons were not altered as demonstrated by Cux-1 immunostaining. These data indicate that the absence of SELENOT impairs the migration of early-born neuroblasts in the neocortex and alters their positioning in the different deep cortical layers. The proper establishment of upper cortical neurons, which are late-born neuroblasts, could be due to a compensatory mechanism in the neocortical environment as we have previously shown

that such adaptation mechanisms operate during late development of Nes-Cre/SELENOT<sup>f/f</sup> adult mice (Castex et al., 2016). These findings add to the list of genes whose expression is essential for the laminar organization of the neocortex, such as the extracellular matrix protein reelin or the glutamate transporter whose disruption in mice provokes neuroblast stagnation in the ventricular layer, accompanied by a dis-organisation of radial glial fibres (Caviness VSJr., 1982; Matsugami et al., 2006; Boyle et al., 2011). It is interesting to note that reelin binds the apolipoprotein E receptor 2 (ApoER2) in neurons (Hiesberger et al., 1999; Scharpf et al., 2007; Hirota et al., 2018), which also serves as a receptor for SELENOP (a selenium transporter to the brain) indicating a possible implication of selenoproteins in important mechanisms involved in cortical lamination. Besides, adhesion molecules such as N-glycosylated integrins whose synthesis could be impacted by SELENOT deficiency (Pothion et al., 2020), also leads to cerebral laminar alteration in mice (Anton et al., 1999; Janik et al., 2010). The abnormalities observed in adult SELENOT KO mice on the one hand, i.e. mispositioning of deep cortical neurons, and after IUE in embryos on the other hand, i.e. abnormal neuroblast polarity, and alterations of radial glial scaffolding and deep neuron positioning, strongly suggest the importance of
<figure><img src="image_8.png" /><figcaption>**Fig. 7.** Neurons of deep cortical layers have positioning defects in adult brain SELENOT-deficient mice. a) Representative images of the neocortex of adult Cre recombinase-expressing WT (Nes-Cre/SELENOT<sup>+/+</sup>) or mutant (Nes-CRE/SELENOT<sup>fl/fl</sup>) mice, labeled with CTIP2, a marker of deep cortical neurons, showing more CTIP2-positive neurons in C6 layer of Nes-Cre/SELENOT<sup>fl/fl</sup> mice compared to Nes-Cre/SELENOT<sup>+/+</sup>, and in contrast, less CTIP2-positive neurons in C5 and C4 layers. Cell nuclei were stained with DAPI (blue). b) Quantification of CTIP2-positive neurons in each cortical layer indicates a 17% increase in C6 and a 5% and 23% decrease in C4 and C5, respectively, in Nes-Cre/SELENOT<sup>fl/fl</sup> compared to Nes-Cre/SELENOT<sup>+/+</sup> neocortex. c) Representative images of the neocortex of adult Cre recombinase-expressing WT (Nes-Cre/SELENOT<sup>+/+</sup>) or mutant (Nes-CRE/SELENOT<sup>fl/fl</sup>) mice, labeled with Cux-1, a marker of upper cortical neurons, showing no positioning difference of Cux-1-expressing cells, as confirmed by quantification of Cux-1-positive neurons in the different cortical layers (d). Data are presented as mean ± SEM (n = 3), \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 using Unpaired t test. C2/3/4/5/6, cortical layer 2/3/4/5/6; WM, white matter. Scale bar 50 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

SELENOT for the proper laminar organization of the neocortex by modulating these processes. Since the localization of neurons in cortical layers is correlated to their identity and connectivities with cortical and subcortical structures (Kwan et al., 2012), it is conceivable that the hyperactive phenotype observed in adult Nes-Cre/SELENOT<sup>fl/fl</sup> mice that we previously reported (Castex et al., 2016) could be ascribed to neuron mispositioning induced by alteration of neuroblast migration.

## Conclusion

Our present data show for the first time that SELENOT is an essential actor in the neocortex required during embryogenesis for cortical lamination, but probably also during the whole mouse lifespan to protect this cerebral structure. We demonstrated the importance of SELENOT during corticogenesis for proper neuroblast migration and

positioning. SELENOT could exert its effects by regulating, via redox mechanisms, different cellular processes necessary for proliferation, migration and differentiation of neuroblasts, including maturation of adhesive molecules and scaffolding of radial glial cells.

Ethics. All animal experimental procedures were reviewed by our regional ethics committee, and approved by the French Higher Education and Research Ministry (Authorization No. 25444, No. 6637 and No. 16178).

Declaration of AI use. We have not used AI-assisted technologies in creating this article.

## Funding

This work was supported by INSERM, the University of Rouen Normandy, the Regional Council of Normandy, DOIT, PHEDERCPG and 3R
FEDER programs, and the French Ministry of Higher Education and Research. Europe gets involved in Normandy through the FEDER program.

## CRediT authorship contribution statement

**Emmanuelle Carpentier:** Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. **Anthony Falluel-Morel:** Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Lisa Brunet:** Methodology, Investigation. **Magalie Bénard:** Methodology. **David Alexandre:** Methodology, Formal analysis. **David Godefroy:** Methodology. **Ben Yamine Mallouki:** Methodology. **Loubna Boukhzar:** Methodology. **Arnaud Arabo:** Resources. **Youssef Anouar:** Writing – original draft, Validation, Supervision, Project administration, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

Images were acquired in PRIMACEN, the cell imaging platform of Normandy.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.neuroscience.2025.09.015.

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