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Anastassia Stoykova
Anastassia Stoykova
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Phone:+49 551 201-1710, -1469Fax:+49 551 201-1504

Anastassia Stoykova

Fig. 4: Conditional activation of Pax6 in the developing cortex of transgenic mice causes progenitor apoptosis. Zoom Image
Fig. 4: Conditional activation of Pax6 in the developing cortex of transgenic mice causes progenitor apoptosis.

The emerging view suggests that differentiation from stem cells through fated progenitors to differentiated neurons and glial cells proceeds through sequence of stages, associated with timely specific expression of TFs in distinct pathways. We have demonstrated that Pax6 is an intrinsic determinant of the radial glial progenitors, which nowdays are known to produce both neuronal and glial cells. By using Cre-LoxP based approaches for conditional recombination in ES cells and transgenic technology, we have generated mouse lines allowing conditional expression of Pax6 in cortical progenitors. The results from the performed analysis indicated that upon overexpression of Pax6, the outcome (enhanced neurogenesis, misregulation of mitotic cycle or cell death) depends on the endogenous Pax6 dosage in the progenitors of distinct cortical domains (Fig. 4).

Fig. 5: Selective cortical layering abnormalities and behavioral deficits in cortex-specific Pax6 knockout mice. Zoom Image
Fig. 5: Selective cortical layering abnormalities and behavioral deficits in cortex-specific Pax6 knockout mice.

Using a similar approach, we found that conditional deletion of Pax6 in cortical progenitors, the adult cortex contains an excess of lower layer (L5) neuronal lineages, generated by the early progenitors, while upper neuronal layers (L4-L2) are almost completely abrogated (Fig. 5). Mechanistically, we found that by regulating the progenitor exit from the mitotic cycle, Pax6 controls the output of the early born lower layer neurons, keeping thereby the progenitor pool for generation of neurons with upper layer identity late in development.

In vivo MRI analysis of the brain of cortex-specific Pax6 knock-out mice (Pax6cKO) revealed disturbances in the cortical organization, indicating a role of Pax6 in neuronal fiber connectivity (Fig. 6). Because of detected selective shrinkage of the motor in expense of the somatosensory and visual areas in the Pax6-deficient Small eye (Sey/Sey) embryos, it was assumed that the cortical areal identity is controlled by the TF Pax6. Thalamocortical tracing in adult conditional Pax6cKO mice revealed, however, that while the graded Pax6 expression in the germinal zone influences the molecular regionalization of the cortex, it does not disturb the establishment of the areal identity (Fig. 7). Furthermore, the generated novel Pax6cKO mouse line showed sensorimotor and cognitive disabilities, similar to deficiencies of aniridia patients with PAX6 defect, thus representing the first mouse model for this human genetic disorder.

Fig. 7: Altered molecular regionalization and normal thalamocortical connections in cortex-specific Pax6 knock-out mice. Zoom Image
Fig. 7: Altered molecular regionalization and normal thalamocortical connections in cortex-specific Pax6 knock-out mice.
Fig. 6: In vivo MRI of altered brain anatomy and fiber connectivity in adult Pax6 deficient mice. Zoom Image
Fig. 6: In vivo MRI of altered brain anatomy and fiber connectivity in adult Pax6 deficient mice.

Fig. 8: TRIM11 modulates the function of neurogenic transcription factor Pax6 through ubiquitin proteasome system. Zoom Image
Fig. 8: TRIM11 modulates the function of neurogenic transcription factor Pax6 through ubiquitin proteasome system.

Accumulating evidence indicates that the dosage of Pax6 protein in progenitor cells has a pivotal role in corticogenesis and forebrain patterning. Recently, we demonstrated an important role for Trim11 in ubiquitination and proteosome-mediated degradation of Pax6 protein. Our data indicate that an autoregulatory feedback loop between Trim11 and Pax6 exists that assures maintenance of Pax6 protein at a physiological level in cortical progenitors, having an essential role for a normal neurogenesis (Fig. 8).

Fig. 9:
<p>Upper row: In situ hybridization analyses revealed expression of <i>Scratch2 (Scrt2)</i> at the border subventricular / intermediate zone of E15.5 embryonic cortex, mostly confined to differentiating Scrt2+/NeuroD1+ cells. Middle row: Overexpression of <i>Scrt2 </i>in cortical progenitors of generated transgenic mice <i>(JoScrt2;Emx1Cre)</i> causes reduction of cortical thickness as compared to controls <i>(JoScrt2),</i> predominantly affecting the generation of neurons with upper layer identity as illustrated through IHC with Cux1 antibody. Low row: Embryo brains at E14.5 were electroporated by <i>in utero</i> electroporation with GFP-expression plasmids as indicated, and analyzed at E18.5. Simultaneous forced expression of <i>Scrt2 </i>together with <i>Rnd2</i> rescued the delayed migration of neurons towards the cortical plate (CP), detected under <i>Scrt2</i> overexpression.</p> Zoom Image
Fig. 9:

Upper row: In situ hybridization analyses revealed expression of Scratch2 (Scrt2) at the border subventricular / intermediate zone of E15.5 embryonic cortex, mostly confined to differentiating Scrt2+/NeuroD1+ cells. Middle row: Overexpression of Scrt2 in cortical progenitors of generated transgenic mice (JoScrt2;Emx1Cre) causes reduction of cortical thickness as compared to controls (JoScrt2), predominantly affecting the generation of neurons with upper layer identity as illustrated through IHC with Cux1 antibody. Low row: Embryo brains at E14.5 were electroporated by in utero electroporation with GFP-expression plasmids as indicated, and analyzed at E18.5. Simultaneous forced expression of Scrt2 together with Rnd2 rescued the delayed migration of neurons towards the cortical plate (CP), detected under Scrt2 overexpression.

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The cortical neurons are generated through the modes of direct and indirect neurogenesis, the first one producing neurons directly from radial glial cells (RGPs), while the second one through generated intermediate progenitors (IPs) that amplify particular neuronal fates. Our recent studies indicate that conditional activation of TF Scratch2 in cortical progenitors of transgenic mice promotes neuronal differentiation by favoring the direct mode of neurogenesis of RGP, at the expense of IP generation. Moreover, Scratch2 controls neuronal migration by negatively regulating the transcriptional activation of bHLH TFs Ngn2/NeuroD1 on E-box-containing target genes, including Rnd2, recently identified by other groups as a major effector of migrational ability of the neurons (Fig. 9).

 
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