Early loss of Scribble affects cortical development and interhemispheric connectivity resulting in psychomotor dysregulation

Neurodevelopmental disorders arise from combined defects in processes including cell proliferation, differentiation, migration and commissure formation. The evolutionarily conserved tumor-suppressor protein Scribble (Scrib) serves as a nexus to transduce signals for the establishment of apicobasal and planar cell polarity during these processes. Human SCRIB gene mutations are associated with neural tube defects and this gene is located in the minimal critical region deleted in the rare Verheij syndrome. In this study, we generated brain-specific conditional cKO mouse mutants and assessed the impact of the Scrib deletion on brain morphogenesis and behavior. We showed that embryonic deletion of Scrib in the telencephalon leads to cortical thickness reduction (microcephaly) and alteration of interhemispheric connectivity (corpus callosum and hippocampal commissure agenesis). We correlated these phenotypes with the identification of novel roles for Scrib, both cell- and non-cell-autonomous, on neuronal migration and axonal guidance respectively. Finally, we show that Scrib cKO mice have psychomotor deficits such as locomotor activity impairment and memory alterations. Altogether, we show that Scrib is essential for early brain development and that the outcomes of its brain-specific disruption support a direct or indirect participation of Scrib to neurodevelopmental pathologies.


Introduction
The mammalian brain, seat of cognitive and behavioral processing, is the result of numerous, complex but coordinated mechanisms of development. Patients with caudal cerebral cortex (~25%) (Fig 3B). This decrease was observed throughout the 135 cingulate (Cg), motor (M), primary somatosensory (S1) and secondary somatosensory 136 (S2) cortices and was maintained in adults (data not shown). This phenotype was 137 absent in more rostral regions of Emx1-Scrib -/-cKO brains (Fig 3C). FoxG1-Scrib -/-138 mutant brains displayed a similar but more severe reduction of cortical area and 139 thickness along the entire rostrocaudal axis (Fig S2A-D). These results on cortical the 140 cortical size reduction observed after early Scrib loss most probably arise from a 141 combination of cell fate and migration defects.

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Considering that cKOs mice had impaired hippocampal and cortical connectivity,  genotypes. Finally, hippocampus-dependent context and amygdala-dependent tone 254 associative memory was assessed by using classical fear conditioning paradigm in 255 which the animals have to associate environmental cues to an electric shock (Fig 8C).

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Two different groups of Emx1-Scrib -/and control mice were re-exposed to the same environment 24 hours or 7 days after training, respectively showing the recent (24h) and 258 the remote (7 days) contextual fear memory (Fig 8D-E in mutants with embryonic Scrib deletion is most likely the result of a combination of 290 disrupted neurogenesis, cell fate and migration processes, as deficits in any of these 291 critical mechanisms can participate in brain malformation 2 . Our data show that this 292 phenotype arises from a depletion in the neuronal lineage, as observed by a decrease 293 in CuxI-and Satb2-positive (layer II/III) CPNs, but also in Ctip2-positive (layer V) CPNs.

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The deletion of Scrib is accompanied by cortical layering defects with mispositioned 295 neurons from layer V, suggesting migration defects that were validated by acute Scrib 296 knockdown. This multifactor effect of Scrib is consistent with its expression profile. It is 297 expressed in the cerebral cortex, both in glutamatergic neurons and in the radial glia, 298 also supporting a role in migration. However, we also observed Scrib enrichment in the 299 VZ and SVZ and we cannot rule out the possibility that it can contribute to asymmetric 300 cell division (ACD) and cell fate determination, as observed in other systems 6,7 .

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The other main phenotype we observed after early Scrib deletion was the CC  Exploration of cognitive performance shows that Emx1-Scrib -/display memory deficit for 342 a cortex-dependent remote-cued fear memory recall. The CC primary function is to 343 integrate motor, sensory, and cognitive activity between the two hemispheres 3 . The   Fianium white laser, a sCMOS Andor camera, and a 0.5 NA 2X objective with a deeping 468 lens. A zoom from 0.63 to 6.3 could be applied. Images and 3D reconstruction were 469 analyzed with Image J and Imaris software. animals were employed for behavioral testing; when the same cohort was used for 501 multiple tests, the most stressful assays were performed last, to minimize between-test 502 interference. To look for behavioral abnormalities mice were tested in activity cages (to 503 measure locomotor activity); in elevated plus maze, open field and Y-maze (to measure 504 exploratory activity and anxiety-like behavior); in rotarod, hot plate, beam walking and 505 grid handling test (to measure sensory-motor activity); and in the fear conditioning test 506 maze (to measure early and remote contextual end cued memory performance). All              hippocampus, but also from the ganglionic eminence that will give rise to GABAergic 915 interneurons. In Emx1-cre mice, the Cre recombinase is expressed later as compared 916 with FoxG1-Cre mice, starting as early as E10.5 and only in the dorsal telencephalon 6 .  anesthetized using 4 % isoflurane in an anesthesia induction chamber, maintained with 992 2 % isoflurane with an anesthetic mask and injected before surgery with buprenorphine.

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Mice were subjected to abdominal incision; uterine horns were exposed and E14.5 994 embryos were placed on humidified gauze pads. Plasmid DNA was purified on Qiagen time of 15 minutes was allowed between each trial. The end of a trial was considered 1018 when mice were falling off the rod. Latency to fall was recorded for each trial. The 1019 average latency was calculated for each testing day.