C3G TUBE PDF

Development of the nervous system Abstract During cerebral cortex development, pyramidal neurons migrate through the intermediate zone and integrate into the cortical plate. These neurons undergo the multipolar—bipolar transition to initiate radial migration. While perturbation of this polarity acquisition leads to cortical malformations, how this process is initiated and regulated is largely unknown. Here we report that the specific upregulation of the Rap1 guanine nucleotide exchange factor, RapGEF2, in migrating neurons corresponds to the timing of this polarity transition.

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Development of the nervous system Abstract During cerebral cortex development, pyramidal neurons migrate through the intermediate zone and integrate into the cortical plate. These neurons undergo the multipolar—bipolar transition to initiate radial migration. While perturbation of this polarity acquisition leads to cortical malformations, how this process is initiated and regulated is largely unknown. Here we report that the specific upregulation of the Rap1 guanine nucleotide exchange factor, RapGEF2, in migrating neurons corresponds to the timing of this polarity transition.

Thus, the specific expression and Cdk5-dependent phosphorylation of RapGEF2 during multipolar—bipolar transition within the intermediate zone are essential for proper neuronal migration and wiring of the cerebral cortex. Download PDF Introduction The migration of neurons from their birthplace to their final destination is fundamental to the architectural formation and functional wiring of the nervous system.

In the mammalian neocortex, most pyramidal neurons are appropriately positioned in distinct cortical layers via three coordinated migration modes: multipolar migration, glia-guided locomotion and somal translocation 1 , 2 , 3.

In particular, cortical neurons first undergo multipolar migration in the lower intermediate zone before they take on a bipolar morphology in the upper intermediate zone to initiate glia-guided locomotion and radially migrate through the cortical plate. Once the migrating neurons reach the marginal zone, they position their cell bodies into their final location by somal translocation 1.

The multipolar—bipolar transition notably serves as a turning point to initiate locomotion. Perturbation of this transition disables glia-guided locomotion, which leads to cortical malformations such as periventricular heterotopia, subcortical band heterotopia and lissencephaly 4. In turn, these conditions are associated with neuropsychiatric disorders such as epilepsy and schizophrenia 4 , 5 , 6.

Although several Cdk5 substrates regulate glia-guided locomotion via leading process dynamics for example, Pak1 and p27Kip1 10 , 11 and nucleokinesis for example, Nudel, DCX and FAK 12 , 13 , 14 , recent evidence suggests that Cdk5 may begin to exert its function in multipolar—bipolar transition to induce locomotion Nonetheless, the in vivo downstream target s that mediates Cdk5 function in multipolar—bipolar transition remains unclear. Rap1 signalling is involved in neuronal migration, and is implicated to be regulated by Cdk5 ref.

Other domains appear to regulate its activity, stability and localization 22 , 23 , Importantly, RapGEF2-deficient mice exhibit heterotopic bands in the subcortical area 25 , implicating its role in early brain development. Furthermore, RapGEF2-mediated Rap1 activation is implicated in other processes of neuronal morphogenesis 26 , Despite the potential significance of RapGEF2 in brain development, surprisingly little is known about its regulation in the developing brain.

It is also unclear whether the two RapGEFs, C3G and RapGEF2 have redundant or specific function in different cellular events during brain development, and how their activity is precisely controlled. The present study demonstrates the cell-autonomous function of RapGEF2 in neuronal migration during cortical development.

In contrast to other neuronal migration regulators expressed ubiquitously in the intermediate zone, RapGEF2 is preferentially found in the radial migration zone and not the multipolar migration zone.

Importantly, the activity of RapGEF2 increases upon phosphorylation by Cdk5, whose kinase activity is also largely restricted to the radial migration zone. Thus, the precise control of RapGEF2 activity through its specific expression and Cdk5-dependent phosphorylation is critical for proper neuronal and cortical circuit assembly. Results RapGEF2 is developmentally regulated in the neocortex To study the function of RapGEF2 in neuronal migration, we first examined its spatial and temporal expression profiles in the developing mouse cortex Fig.

In western blot analyses of mouse cortex extracts at different developmental stages embryonic day E 12 to postnatal day P 5 , RapGEF2 was barely detectable in E12 mouse cortices. Its expression was upregulated during the critical stages of neuronal migration in the developing neocortex and remained high thereafter Fig. To detect the spatial expression pattern of RapGEF2 in the cortical wall of developing mouse brains, we examined coronal cortical sections from mouse embryos collected at E12, E15 and E RapGEF2 was barely detected at E12 neocortex, which mainly comprises neural progenitor cells.

At E15 and E17, when robust neuronal migration occurs, RapGEF2 expression was prominently upregulated in nascent migrating neurons located in the upper intermediate zone. In utero electroporation was used to further visualize migrating neurons in the developing cortex, revealing that RapGEF2 was upregulated precisely when migrating neurons obtained a bipolar morphology in the upper intermediate zone Fig. These findings prompted us to investigate whether the dynamic regulation of RapGEF2 during multipolar—bipolar transition is essential for migrating neurons to pass through the intermediate zone and integrate into their destined cortical layers.

Actin served as the loading control. See full-length blots in Supplementary Fig. Double-headed arrows, upper intermediate zone u-IZ. Representative E The experiment was repeated for at least three times. Full size image RapGEF2 regulates neuronal migration to the cortical plate The restrictive expression of RapGEF2 in the upper intermediate zone during cortical development prompted us to examine its functional importance in neuronal migration by acutely abrogating its expression in mouse embryos through in utero electroporation.

RapGEF2 knockdown disrupted the migration of projection neurons during neocortical development. Detailed examination of cell morphology showed that most control neurons had a characteristic bipolar morphology with one major leading process oriented towards the cortical plate.

Conversely, most RapGEF2-knockdown neurons arrested in the intermediate zone failed to undergo multipolar—bipolar transition and exhibited abnormal morphologies with either multiple short processes or no neuronal process Fig. Figure 2: RapGEF2 regulates neuronal migration to the cortical plate. Asterisks indicate representative neurons in each group. Error bars indicate the s. These results strongly suggest that RapGEF2 is required for multipolar—bipolar conversion and neuronal migration.

Furthermore, the observed phenotype on RapGEF2 suppression is not the secondary effect of defects in other developmental processes. Because neurons generated from E14 integrate into the upper layers that is, layers II—IV of the cerebral cortex, we speculated that RapGEF2 controls the migration of newborn neurons that assemble to form different cortical layers. These RapGEF2-suppressed neurons were similarly positioned within the intermediate zone and failed to enter the cortical plate 3 days after electroporation Supplementary Fig.

This result suggests that RapGEF2 function is essential for the multipolar—bipolar transition and migration of neurons from different cortical layers. There is a neurogenic gradient between the lateral cortex and the dorsomedial cortex that is, the former is older than the latter.

Accordingly, electroporation of the lateral cortex at E12 labels those neurons that pass through the multipolar migration phase and mainly use locomotion 2 , 28 , whereas electroporation of the dorsomedial cortex labels the neurons that only use somal translocation RapGEF2 regulates multipolar—bipolar transition Before radial locomotion in the cortical plate, migrating neurons undergo a transient multipolar migration phase in the intermediate zone in which they exhibit random active neurite extension and retraction, and subsequently acquire a radially oriented bipolar morphology.

Control neurons exhibited active neurite extension and retraction, and eventually acquired a radially oriented leading process towards the pial surface Fig. Conversely, RapGEF2-suppressed neurons exhibit impaired bipolar transition and reduced migration speed Fig.

Nevertheless, RapGEF2 knockdown does not regulate neurite dynamics, because neither the number of neurite extension or retraction events nor average lifetime of extended neurites was affected Fig. Thus, these observations indicate that RapGEF2 regulates the formation of a radially oriented leading process, probably through a cellular mechanism besides neurite dynamics. Figure 3: RapGEF2 is essential for multipolar—bipolar transition. Red arrowheads, neurites extending from cell bodies.

White arrowhead, swelling of leading process of a control neuron. Full size image RapGEF2 knockdown causes accumulation of ectopic neurons To determine whether defective migration induced by RapGEF2 shRNA temporarily or permanently affects cortical lamination, we examined the cerebral cortex 6 days post electroporation at P2.

Most control neurons migrated up to the cortical plate and subsequently settled in cortical layers II—IV Fig. Meanwhile, most RapGEF2-suppressed neurons remained accumulated in the intermediate zone, leading to the malformation of an ectopic neuronal layer beneath the cortical plate positive for Cux1, a layer II—IV marker Fig. Figure 4: RapGEF2 knockdown causes accumulation of ectopic neurons.

Arrows denote the heterotopic band of arrested neurons. Asterisks indicate representative neurons. Error bars indicate s. More than cells were analysed from three brains in each group. Full size image To determine the long-term consequences of RapGEF2 depletion at E14, we examined the mouse cerebral cortex at P20, when neuronal migration is normally completed.

RapGEF2 knockdown strikingly resulted in a heterotopic band of neurons in the subcortical white matter, which corresponds to the intermediate zone in the developing cortex Fig. Thus, neurons with suppressed RapGEF2 expression permanently failed to migrate up and integrate into the proper cortical layers. Further characterization of these trapped RapGEF2-knockdown neurons revealed a defective phenotype similar to that observed during earlier developmental stages E17 and P2 , that is, a severely perturbed morphology characterized by a distorted cell body shape and abnormal dendritic morphogenesis.

Exit from multipolar phase requires RapGEF2 activity The small GTPase Rap1 has been suggested to play important roles in multiple steps of neuronal migration, including multipolar—bipolar transition and somal translocation 17 , However, the spatiotemporal regulation of Rap1 activity during distinct migration phases remains unclear.

As mentioned earlier, RapGEF2 knockdown resulted in neuronal mislocalization and arrest in the multipolar cell phase Figs 2a,c,d and 5a—c. Meanwhile, C3G knockdown neither impaired the bipolar transition nor delayed neuronal migration into the cortical plate at E17 Fig. These results are concordant with those of a recent study suggesting that C3G pathway is required for terminal translocation Together, these findings indicate that neurons specifically require RapGEF2 but not C3G to exit the multipolar cell phase and migrate into the cortical plate.

Thus, although both GEFs can regulate Rap1 activity, they play specific roles in different cellular events during neuronal migration. Thus, the replenishment of active Rap1 protein indeed restores normal neuronal migration in RapGEF2-knockdown cortices.

These findings collectively show that RapGEF2-dependent Rap1 activation during bipolar transition is a critical molecular event that safeguards proper neuronal migration in vivo.

In an independent study, we attempted to identify novel Cdk5 substrates using mass spectrometry and revealed that a phosphopeptide with amino-acid sequence derived from RapGEF2 is decreased in the brains of Cdk5-conditional knockout mice 33 Ip et al.

The phosphopeptide identified by the phosphoproteomic analysis is shown. The three proline-directed serine sites are highlighted. Full size image To determine the specificity and spatial expression pattern of phospho-RapGEF2 in the mouse cortex during neuronal migration, we stained coronal cortical sections from mouse embryos collected at E15 with phospho-RapGEF2 antibody.

The staining pattern of phospho-RapGEF2 was similar to that of total RapGEF2 protein, showing restrictive signals in the upper intermediate zone and cortical plate as well as co-localization with neuronal marker Tuj1 Fig.

The phospho-staining pattern was abolished when the phospho-RapGEF2 antibody was pre-absorbed with the phosphopeptide antigen; this indicates that the antibody specifically recognizes phospho-RapGEF2 Supplementary Fig.

In addition, p35, the major Cdk5 activator during brain development, exhibited a similar restrictive distribution pattern as that of phospho-RapGEF2 in the developing cerebral cortex Fig. Importantly, immunohistochemical analysis of E15 cortical sections from wild-type and Cdk5-deficient mice showed that RapGEF2 phosphorylation at Ser was barely detected in the intermediate zone or cortical plate of Cdk5-deficient cortices Fig.

RapGEF2 WT or mutants in which the phosphorylation site was changed to alanine or glutamate to mimic nonphosphorylated or phosphorylated states, respectively, were expressed in HEKT cells. Mutation of Ser to alanine resulted in a significantly lower level of active Rap1 Fig. To determine whether Cdk5 regulates Rap1 activation in vivo during the developmental stage of neuronal migration, we measured Rap1 activity in E18 Cdk5-knockout brains. Concordant with our hypothesis, active Rap1 levels were significantly lower in the cortex of Cdk5-deficient mice than their wild-type littermates Fig.

These results collectively suggest that Cdk5 is involved in Rap1 activation during cortical development. Full size image As mentioned above, the GEF activity of RapGEF2 is specifically required for multipolar—bipolar transition and neuronal entry into the cortical plate during neuronal migration.

To study the regulatory role of the Cdk5-mediated RapGEF2 phosphorylation in neuronal migration, various RapGEF2 mutants were re-expressed in RapGEF2-depleted cortices and their effects on the restoration of proper neuronal migration were examined Fig.

However, the re-expression of the phosphodeficient mutant of RapGEF2, SA, which exhibited reduced Rap1 activity in vitro, failed to rescue the defective multipolar—bipolar transition or migration of cortical neurons caused by RapGEF2 depletion Fig. In many cell types, Rap1 regulates the trafficking of cell surface molecules between endosomes and the plasma membrane 35 , Interestingly, recent evidence suggests that Rap1 regulates N-cadherin localization in cortical neurons during neuronal migration Neurons were examined by immunocytochemical analysis 2 days later.

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