ex-utero-electroporation-whole-hemisphere-explants-simple

<em>Ex utero</em> Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Lissencephaly, which means 'smooth cortex', is caused by defective neuronal migration during development of the cerebral cortex and has devastating clinical consequences. 'Classical' lissencephaly seems to reflect mutations in regulators of the microtubule cytoskeleton, whereas 'cobblestone' lissencephaly is caused by mutations in genes needed for the integrity of the basal lamina of the central nervous system. Reelin, which is mutated in a third type of lissencephaly, may represent a unifying link because it encodes an extracellular protein that regulates neuronal migration and may also regulate the microtubule cytoskeleton.

Smooth, rough and upside-down neocortical development.

Lissenzephalie, was &#39;glatte Rinde&#39; bedeutet, wird verursacht durch fehlerhafte neuronale Migration w&auml;hrend der Entwicklung der Gro&szlig;hirnrinde und hat verheerende klinische Folgen. &#39;Klassischen&#39; Lissenzephalie scheint Mutationen im Regulatoren des Microtubule Zytoskeletts, widerspiegeln, w&auml;hrend &#39;Pflasterstein&#39;-Lissenzephalie durch Mutationen in Genen, die notwendig f&uuml;r die Integrit&auml;t der Basallamina des zentralen Nervensystems verursacht wird. Reelin, die in einer dritten Art von Lissenzephalie mutiert ist m&ouml;glicherweise dar eine einigende Verbindung weil es eine extrazellul&auml;re Protein kodiert, das regelt der neuronalen Migration und k&ouml;nnen auch Regeln, die Microtubule Cytoskeleton.

Glatte, rauhe und Kopf neokortikalen Entwicklung.

Reelin and Disabled 1 (Dab1) are essential for positioning migrating neurons in the developing neocortex. Cell-autonomous RNA interference-mediated suppression of Dab1 in migrating neurons destined for layer 2/3 shifted the median position of these cells to deeper positions within the cortex. At the time of migration arrest [embryonic day 20 (E20) to E21], Dab1-suppressed cells were underrepresented in the upper approximately 40 microm of the cortex compared with controls, suggesting that Dab1 is essential for somal translocation through the cell-dense cortical plate. Closer examination of the morphology of Dab1-suppressed neurons at E20 revealed simplified leading processes that are less likely to contact the marginal zone (MZ), in which high levels of Reelin are expressed. Examination of Dab1-suppressed cells 3 d later (postnatal day 2) revealed simplified dendrites that are also less likely to contact the MZ. These data reveal a cell-autonomous role of Dab1 in dendritogenesis in the neocortex and suggest that remodeling of the leading process of a migrating neuron into a nascent dendrite by Reelin/Dab1 signaling plays an important role in cell positioning.

Impaired neuronal positioning and dendritogenesis in the neocortex after cell-autonomous Dab1 suppression.

Reelin und Behinderte 1 (Dab1) sind wesentlich f&uuml;r die Positionierung der Migration von Neuronen in Entwicklungsl&auml;ndern Neocortex. Zelle-autonome RNA Interferenz-vermittelte Unterdr&uuml;ckung der Dab1 in Migration von Neuronen, die bestimmt f&uuml;r Schicht 2/3 die mittlere Position dieser Zellen zur tieferen Positionen innerhalb der Hirnrinde verschoben. Zum Zeitpunkt der Migration Verhaftung [embryonalen 20.Tag (E20) zu E21] wurden Dab1 unterdr&uuml;ckt Zellen in die obere etwa 40 Microm der Hirnrinde, im Vergleich zu Kontrollen, die darauf hindeutet, dass Dab1 f&uuml;r somal Translokation durch die Zelle-Dichte kortikale Platte wichtig unterrepr&auml;sentiert. N&auml;herer Untersuchung der Morphologie der Dab1 unterdr&uuml;ckt Neuronen bei E20 offenbart vereinfachte f&uuml;hrenden Prozesse, sind weniger wahrscheinlich die Randzone (MZ), Kontakt, in denen hohen Konzentrationen von Reelin ausgedr&uuml;ckt werden. Pr&uuml;fung Dab1 unterdr&uuml;ckt Zellen 3 d sp&auml;ter (postnatale 2.Tag) ergab vereinfachte Dendriten, die auch weniger wahrscheinlich, die MZ zu kontaktieren. Diese Daten zeigen eine Zelle-autonome Rolle der Dab1 in Dendritogenesis in der Gro&szlig;hirnrinde und schlagen vor, da&szlig; die Umgestaltung des f&uuml;hrenden Prozesses einer Migration von Nervenzelle in einer aufkeimenden Dendrit durch das signalisieren Reelin/Dab1 spielt eine wichtige Rolle in der Zelle positionieren.

Wertminderung neuronalen Positionierung und Dendritogenesis in der Gro&szlig;hirnrinde nach Zelle-autonome Dab1 Unterdr&uuml;ckung.

Overexpression of beta-catenin, a protein that functions in both cell adhesion and signaling, causes expansion of the cerebral cortical precursor population and cortical surface area enlargement. Here, we find that focal elimination of beta-catenin from cortical neural precursors in vivo causes premature neuronal differentiation. Precursors within the cerebral cortical ventricular zone exhibit robust beta-catenin-mediated transcriptional activation, which is downregulated as cells exit the ventricular zone. Targeted inhibition of beta-catenin signaling during embryonic development causes cortical precursor cells to prematurely exit the cell cycle, differentiate into neurons, and migrate to the cortical plate. These results show that beta-catenin-mediated transcriptional activation functions in the decision of cortical ventricular zone precursors to proliferate or differentiate during development, and suggest that the cell-autonomous signaling activity of beta-catenin can control the production of cortical neurons and thus regulate cerebral cortical size.

Cell-autonomous beta-catenin signaling regulates cortical precursor proliferation.

Mutations, toxic insults and radiation exposure are known to slow or arrest the migration of cortical neurons, in most cases by unknown mechanisms. The movement of migrating neurons is saltatory, reflecting the intermittent movement of the nucleus (nucleokinesis) within the confines of the plasma membrane. Each nucleokinetic movement is analogous to a step. Thus, average migration speed could be reduced by lowering step frequency and/or step distance.

Comparison of slow and fast neocortical neuron migration using a new in vitro model.

The secreted ligand Reelin is thought to regulate the translocation and positioning of prospective layer 6 (L6) neurons into the preplate, a plexus of neurons overlying the ventricular zone. We examined wild type and Reelin-deficient cortices and found that L6 neurons were equivalently positioned beneath the pia during the period of preplate splitting and initial cortical plate (CP) formation. The absence of detectable L6 ectopia in "reeler" cortices at this developmental time point indicates that Reelin-signaling might not regulate L6 neuron migration or gross positioning during preplate splitting. To explore the acute response of L6 neurons to Reelin, subpial injections of Reelin were made into Reelin-deficient explants. Reelin injection caused L6 neurons to orient their nuclei and polarize their Golgi toward the pia while initiating exuberant dendritic (MAP2+) outgrowth within 4 h. This rapid Reelin-dependent neuronal orientation and alignment created CP-like histology without any significant change in the mean position of the population of L6 neurons. Conversely, subplate cells and chondroitin sulfate proteoglycan immunoreactivity were found at significantly deeper positions from the pial surface after injection, suggesting that Reelin partially rescues preplate splitting within 4 h. Thus, Reelin has a direct role in promoting rapid morphological differentation and orientation of L6 neurons during preplate splitting.

Reelin promotes neuronal orientation and dendritogenesis during preplate splitting.

The Reelin ligand regulates a Dab1-dependent signaling pathway required for brain lamination and normal dendritogenesis, but the specific mechanisms underlying these actions remain unclear. We find that Stk25, a modifier of Reelin-Dab1 signaling, regulates Golgi morphology and neuronal polarization as part of an LKB1-Stk25-Golgi matrix protein 130 (GM130) signaling pathway. Overexpression of Stk25 induces Golgi condensation and multiple axons, both of which are rescued by Reelin treatment. Reelin stimulation of cultured neurons induces the extension of the Golgi into dendrites, which is suppressed by Stk25 overexpression. In vivo, Reelin and Dab1 are required for the normal extension of the Golgi apparatus into the apical dendrites of hippocampal and neocortical pyramidal neurons. This demonstrates that the balance between Reelin-Dab1 signaling and LKB1-Stk25-GM130 regulates Golgi dispersion, axon specification, and dendrite growth and provides insights into the importance of the Golgi apparatus for cell polarization.

Reelin and stk25 have opposing roles in neuronal polarization and dendritic Golgi deployment.

Layer 6 cortical neurons require Reelin-Dab1 signaling for cellular orientation, Golgi deployment, and directed neurite growth into the marginal zone.

Schicht 6 kortikale Neuronen erfordern Reelin-Dab1 Signalisierung f&uuml;r zellulare Orientierung, Golgi-Bereitstellung und gesteuerte Neurit Wachstum in der Randzone.

Prenatal ethanol exposure disrupts cortical neurite initiation and outgrowth, but prior studies have reported both ethanol-dependent growth promotion and inhibition. To resolve this ambiguity and better approximate in vivo conditions, we quantitatively analyzed neuronal morphology using a new, whole hemisphere explant model. In this model, Layer 6 (L6) cortical neurons migrate, laminate and extend neurites in an organotypic fashion. To selectively label L6 neurons, we performed ex utero electroporation of a GFP expression construct at embryonic day 13 and allowed the explants to develop for 2 days in vitro. Explants were exposed to (400 mg/dL) ethanol for either 4 or 24 h prior to fixation. Complete 3-D reconstructions were made of >80 GFP-positive neurons in each experimental condition. Acute responses to ethanol exposure included compaction of the Golgi apparatus accompanied by elaboration of supernumerary primary apical neurites, as well as a modest (âˆ¼15%) increase in higher order apical neurite length. With longer exposure time, ethanol exposure leads to a consistent, significant disorientation of the cell (cell body, primary apical neurite, and Golgi) with respect to the pial surface. The effects on cellular orientation were accompanied by decreased expression of cytoskeletal elements, microtubule-associated protein 2 and F-actin. These findings indicate that upon exposure to ethanol, developing L6 neurons manifest disruptions in Golgi apparatus and cytoskeletal elements which may in turn trigger selective and significant perturbations to primary neurite formation and neuronal polarity.

Ethanol-induced disruption of Golgi apparatus morphology, primary neurite number and cellular orientation in developing cortical neurons.

Cortical neurons display dynamic patterns of gene expression during the coincident processes of differentiation and migration through the developing cerebrum. To identify genes selectively expressed by the Eomesâ€‰+â€‰(Tbr2) lineage of excitatory cortical neurons, GFP-expressing cells from Tg(Eomes::eGFP) Gsat embryos were isolated to > 99% purity and profiled.

Article	Year
Smooth, rough and upside-down neocortical development. Current opinion in genetics & development\| PubMed ID: 12076676	2002
Impaired neuronal positioning and dendritogenesis in the neocortex after cell-autonomous Dab1 suppression. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 16467525	2006
Cell-autonomous beta-catenin signaling regulates cortical precursor proliferation. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 17135424	2006
Comparison of slow and fast neocortical neuron migration using a new in vitro model. BMC neuroscience\| PubMed ID: 18534012	2008
Reelin promotes neuronal orientation and dendritogenesis during preplate splitting. Cerebral cortex (New York, N.Y. : 1991)\| PubMed ID: 20064940	2010
Reelin and stk25 have opposing roles in neuronal polarization and dendritic Golgi deployment. Cell\| PubMed ID: 21111240	2010
Layer 6 cortical neurons require Reelin-Dab1 signaling for cellular orientation, Golgi deployment, and directed neurite growth into the marginal zone. Neural development\| PubMed ID: 22770513	2012
Ethanol-induced disruption of Golgi apparatus morphology, primary neurite number and cellular orientation in developing cortical neurons. Alcohol (Fayetteville, N.Y.)\| PubMed ID: 22840816	2012
Hierarchical clustering of gene expression patterns in the Eomes + lineage of excitatory neurons during early neocortical development. BMC neuroscience\| PubMed ID: 22852769	2012

Eric C. Olson

Biography

JoVE Journal Publications

Other Publications