Role of Apc in cortical development

Cerebral cortex development is a complicated process which is not completely understood. There are a lot of signaling interactions during morphogenesis of the cortex. One of the signaling pathways is Wnt. Adenomatous polyposis coli (Apc) is a key regulator of the canonical Wnt pathway. Also Ape is important to transducer signals from the planar cell polarity (PCP) Wnt pathway. This thesis is devoted to the role of Ape protein in the development of the mouse dorsal telencephalon. Apc⁻/⁻ embryos die at gastrulation, therefore Emxlᶜʳᵉ conditional knock-out approach was used to overcome this problem. Cre expression driven by Emxl promoter allows knock-out Apc in the cortex. Current work presents description and possible explanations of Apc functions in the developing cerebral cortex.Conditional knock-out of Apc in the cortex using Emxlᶜʳᵉ leads to severe developmental defects in this region. This shows that Apc is required for normal development of the cerebral cortex. The earliest found defect is nuclear translocation of beta-catenin which is demonstrating that Apc is important to regulate translocation of beta-catenin to the nucleus. This finding supports results of other researchers. This abnormality was found at embryonic day 10.5 (El0.5), which shows that Apc has a controlling function from the beginning of cortical development. Later in development nuclear beta-catenin accumulation becomes more pronounced. Experiments with BatGal reporter mice show that stabilized beta-catenin is able to stimulate the canonical Wnt pathway. Wnt target genes (C-myc, Cyclin Dl) are activated also. However Axin 2 expression is highly up-regulated which reflects negative feedback to the canonical Wnt signaling activation. Polarity of cells is lost from El2.5, which suggests that the cytoskeletal functions of Ape are affected by Ape deletion. Adhesion defect and defect in neuronal processes elongation provide additional evidence of the cytoskeleton disregulation. Therefore, the deletion of Apc leads to over-activation of the canonical Wnt pathway and disregulation of cytoskeleton functioning.Identity of the dorsal telencephalon is unclear in conditional Ape mutant embryos. Expression of Foxgl shows that the mutant dorsal telencephalon loses telencephalic identity from El2.5. There are signs that the mutant dorsal telencephalon expresses markers (Pax3, Wntl) of more caudal regions of the brain. However markers normally expressed in the cerebral cortex (Pax6, Tbr1) are still present. Pax6 is downregulated in the mutant but there are no signs that the pallial subpal 1 ial boundary is compromised. Apical progenitor population is decreased. Decreased Tbr2 expression shows that intermediate progenitor pool is reduced also. Medial regions of the mutant dorsal telencephalon are more affected than lateral possibly due to a gradient in Emxl expression in the cortex. My data show that Ape is important for proper patterning of the cortex probably mostly by antagonizing the canonical Wnt pathway. However a precise mechanism is yet to be elucidated.Cell-cycle investigation revealed that in the absence of Apc, S-phase and cell cycle length remains more or less similar to the control. However the proliferative pool is decreased and most cells of the mutant are blocked in G1 phase. This defect progresses with the age. Aneuploidy was not detected in the mutant cells. The G1 blockade is related to p21 up-regulation. Also apoptosis is increased in the mutant but level of p53 is not changed. My data suggests that apoptosis and p21 expression is stimulated by Wtl expression, which reflects tumour suppression. Thus, Ape deletion leads to defects in maintenance of the size of progenitor pool and regulation of apoptosis

Adenomatous polyposis coli is required for early events in the normal growth and differentiation of the developing cerebral cortex

<p>Abstract</p> <p>Background</p> <p>Adenomatous polyposis coli (Apc) is a large multifunctional protein known to be important for Wnt/β-catenin signalling, cytoskeletal dynamics, and cell polarity. In the developing cerebral cortex, <it>Apc </it>is expressed in proliferating cells and its expression increases as cells migrate to the cortical plate. We examined the consequences of loss of Apc function for the early development of the cerebral cortex.</p> <p>Results</p> <p>We used <it>Emx1</it>^{<it>Cre </it>}to inactivate <it>Apc </it>specifically in proliferating cerebral cortical cells and their descendents starting from embryonic day 9.5. We observed reduction in the size of the mutant cerebral cortex, disruption to its organisation, and changes in the molecular identity of its cells. Loss of Apc leads to a decrease in the size of the proliferative pool, disrupted interkinetic nuclear migration, and increased apoptosis. β-Catenin, pericentrin, and N-cadherin proteins no longer adopt their normal high concentration at the apical surface of the cerebral cortical ventricular zone, indicating that cell polarity is disrupted. Consistent with enhanced Wnt/β-catenin signalling resulting from loss of Apc we found increased levels of TCF/LEF-dependent transcription and expression of endogenous Wnt/β-catenin target genes (<it>Axin2 </it>(<it>conductin</it>), <it>Lef1</it>, and <it>c-myc</it>) in the mutant cerebral cortex. In the <it>Apc </it>mutant cerebral cortex the expression of transcription factors <it>Foxg1</it>, <it>Pax6</it>, <it>Tbr1</it>, and <it>Tbr2 </it>is drastically reduced compared to normal and many cells ectopically express <it>Pax3</it>, <it>Wnt1</it>, and <it>Wt1 </it>(but not <it>Wnt2b</it>, <it>Wnt8b</it>, <it>Ptc</it>, <it>Gli1</it>, <it>Mash1</it>, <it>Olig2</it>, or <it>Islet1</it>). This indicates that loss of Apc function causes cerebral cortical cells to lose their normal identity and redirect to fates normally found in more posterior-dorsal regions of the central nervous system.</p> <p>Conclusion</p> <p>Apc is required for multiple aspects of early cerebral cortical development, including the regulation of cell number, interkinetic nuclear migration, cell polarity, and cell type specification.</p

Adenomatous Polyposis Coli Regulates Axon Arborization and Cytoskeleton Organization via Its N-Terminus

Conditional deletion of APC leads to marked disruption of cortical development and to excessive axonal branching of cortical neurons. However, little is known about the cell biological basis of this neuronal morphological regulation. Here we show that APC deficient cortical neuronal growth cones exhibit marked disruption of both microtubule and actin cytoskeleton. Functional analysis of the different APC domains revealed that axonal branches do not result from stabilized β-catenin, and that the C-terminus of APC containing microtubule regulatory domains only partially rescues the branching phenotype. Surprisingly, the N-terminus of APC containing the oligomerization domain and the armadillo repeats completely rescues the branching and cytoskeletal abnormalities. Our data indicate that APC is required for appropriate axon morphological development and that the N-terminus of APC is important for regulation of the neuronal cytoskeleton

Evidence That Descending Cortical Axons Are Essential for Thalamocortical Axons to Cross the Pallial-Subpallial Boundary in the Embryonic Forebrain

Developing thalamocortical axons traverse the subpallium to reach the cortex located in the pallium. We tested the hypothesis that descending corticofugal axons are important for guiding thalamocortical axons across the pallial-subpallial boundary, using conditional mutagenesis to assess the effects of blocking corticofugal axonal development without disrupting thalamus, subpallium or the pallial-subpallial boundary. We found that thalamic axons still traversed the subpallium in topographic order but did not cross the pallial-subpallial boundary. Co-culture experiments indicated that the inability of thalamic axons to cross the boundary was not explained by mutant cortex developing a long-range chemorepulsive action on thalamic axons. On the contrary, cortex from conditional mutants retained its thalamic axonal growth-promoting activity and continued to express Nrg-1, which is responsible for this stimulatory effect. When mutant cortex was replaced with control cortex, corticofugal efferents were restored and thalamic axons from conditional mutants associated with them and crossed the pallial-subpallial boundary. Our study provides the most compelling evidence to date that cortical efferents are required to guide thalamocortical axons across the pallial-subpallial boundary, which is otherwise hostile to thalamic axons. These results support the hypothesis that thalamic axons grow from subpallium to cortex guided by cortical efferents, with stimulation from diffusible cortical growth-promoting factors

The expression and activity of β-catenin in the thalamus and its projections to the cerebral cortex in the mouse embryo

<p>Abstract</p> <p>Background</p> <p>The mammalian thalamus relays sensory information from the periphery to the cerebral cortex for cognitive processing via the thalamocortical tract. The thalamocortical tract forms during embryonic development controlled by mechanisms that are not fully understood. β-catenin is a nuclear and cytosolic protein that transduces signals from secreted signaling molecules to regulate both cell motility via the cytoskeleton and gene expression in the nucleus. In this study we tested whether β-catenin is likely to play a role in thalamocortical connectivity by examining its expression and activity in developing thalamic neurons and their axons.</p> <p>Results</p> <p>At embryonic day (E)15.5, the time when thalamocortical axonal projections are forming, we found that the thalamus is a site of particularly high β-catenin mRNA and protein expression. As well as being expressed at high levels in thalamic cell bodies, β-catenin protein is enriched in the axons and growth cones of thalamic axons and its growth cone concentration is sensitive to Netrin-1. Using mice carrying the β-catenin reporter <it>BAT-gal </it>we find high levels of reporter activity in the thalamus. Further, Netrin-1 induces <it>BAT-gal </it>reporter expression and upregulates levels of endogenous transcripts encoding β-actin and L1 proteins in cultured thalamic cells. We found that β-catenin mRNA is enriched in thalamic axons and its 3'UTR is phylogenetically conserved and is able to direct heterologous mRNAs along the thalamic axon, where they can be translated.</p> <p>Conclusion</p> <p>We provide evidence that β-catenin protein is likely to be an important player in thalamocortcial development. It is abundant both in the nucleus and in the growth cones of post-mitotic thalamic cells during the development of thalamocortical connectivity and β-catenin mRNA is targeted to thalamic axons and growth cones where it could potentially be translated. β-catenin is involved in transducing the Netrin-1 signal to thalamic cells suggesting a mechanism by which Netrin-1 guides thalamocortical development.</p