Meningeal defects alter the tangential migration of cortical interneurons in Foxc1hith/hith mice

<p>Abstract</p> <p>Background</p> <p>Tangential migration presents the primary mode of migration of cortical interneurons translocating into the cerebral cortex from subpallial domains. This migration takes place in multiple streams with the most superficial one located in the cortical marginal zone. While a number of forebrain-expressed molecules regulating this process have emerged, it remains unclear to what extent structures outside the brain, like the forebrain meninges, are involved.</p> <p>Results</p> <p>We studied a unique <it>Foxc1 </it>hypomorph mouse model (<it>Foxc1^hith/hith</it>) with meningeal defects and impaired tangential migration of cortical interneurons. We identified a territorial correlation between meningeal defects and disruption of interneuron migration along the adjacent marginal zone in these animals, suggesting that impaired meningeal integrity might be the primary cause for the observed migration defects. Moreover, we postulate that the meningeal factor regulating tangential migration that is affected in homozygote mutants is the chemokine Cxcl12. In addition, by using chromatin immunoprecipitation analysis, we provide evidence that the <it>Cxcl12 </it>gene is a direct transcriptional target of Foxc1 in the meninges. Further, we observe migration defects of a lesser degree in Cajal-Retzius cells migrating within the cortical marginal zone, indicating a less important role for Cxcl12 in their migration. Finally, the developmental migration defects observed in <it>Foxc1^hith/hith</it>mutants do not lead to obvious differences in interneuron distribution in the adult if compared to control animals.</p> <p>Conclusions</p> <p>Our results suggest a critical role for the forebrain meninges to promote during development the tangential migration of cortical interneurons along the cortical marginal zone and Cxcl12 as the factor responsible for this property.</p

Oxidative Stress and Regulation of Pink1 in Zebrafish (Danio rerio)

Peer reviewe

Loss of Wdfy3 in mice alters cerebral cortical neurogenesis reflecting aspects of the autism pathology.

Autism spectrum disorders (ASDs) are complex and heterogeneous developmental disabilities affecting an ever-increasing number of children worldwide. The diverse manifestations and complex, largely genetic aetiology of ASDs pose a major challenge to the identification of unifying neuropathological features. Here we describe the neurodevelopmental defects in mice that carry deleterious alleles of the Wdfy3 gene, recently recognized as causative in ASDs. Loss of Wdfy3 leads to a regionally enlarged cerebral cortex resembling early brain overgrowth described in many children on the autism spectrum. In addition, affected mouse mutants display migration defects of cortical projection neurons, a recognized cause of epilepsy, which is significantly comorbid with autism. Our analysis of affected mouse mutants defines an important role for Wdfy3 in regulating neural progenitor divisions and neural migration in the developing brain. Furthermore, Wdfy3 is essential for cerebral expansion and functional organization while its loss-of-function results in pathological changes characteristic of ASDs

In-situ hybridization and q-RT-PCR at 3dpf in wild type fish following treatment with H₂O₂ and H₂O₂+LGR.

<div><p>A. The <i>th2</i> transcript levels significantly decreased in the H₂O₂ treated samples, verified by q-RT-PCR. </p> <p>B. The <i>pink1</i> transcript levels increased significantly in the H₂O₂ treated as compared to wild type samples. </p> <p>C. No significant change in the <i>th1</i> transcript levels was detected between the groups. Only a declining trend was observed.</p> <p>Unpaired t-test (* p-value < 0.05, ** p-value< 0.01).</p> <p>D-F. The expression of <i>th2</i> mRNA is reduced in group 10b of the 7-dpf H₂O₂-treated fish as compared with untreated control fish. In the LGR-treated groups at 8 dpf, the decrease in <i>th2</i> mRNA expression was prevented. The <i>th2</i>-expressing neuron groups 3b, 8b, 9b, 10b are identified according to [26].</p> <p>Scale bar represents 100 μM. <i>th1</i> – tyrosine hydroxylase1, <i>th2</i> – tyrosine hydroxylase 2, <i>pink1</i> – PTEN-induced putative kinase 1.</p></div

The effect of H₂O₂ on GFP and TH1 expression in <i>Tg</i>(<i>pink1:EGFP</i>) larval fish.

<div><p>A-C. GFP-ir at 7 dpf in the zebrafish larvae. GFP-ir increases in the H₂O₂-treated fish (B) as compared to the control fish (A). The effect is rescued in the LGR treated fish (C).</p> <p>D. Mean fluorescence intensity values of the WT, H₂O₂-treated group and the H₂O₂+LGR treated group. A significant change in the H₂O₂-treated group was observed (p < 0.01).</p> <p>E-G. A three-dimensional image to perform volume rendering was constructed using the Imaris MeasurementPro module in Imaris software for the above-mentioned fish at a similar threshold intensity. The changes indicated as the changed total volume of the representative images for control (E), H₂O₂ (F) and LGR-treated fish (G). Scale bar represents 80 μm.</p> <p>H-J. TH-ir is unchanged among the control (H), H₂O₂-treated (I) and H₂O₂ together with LGR (J) groups of larval fish. No change in the pretectal (7) and preoptic, thalamic or pre-tectal nucleus (5,6,11) cell populations was seen. Other visible groups in the olfactory bulb and ventral telencephalic nuclei (1,2), periventricular hypothalamus and posterior tuberculum (13), and the locus coeruleus (14) also displayed no change in TH-ir. </p> <p>Scale bar represents 100 μm. A-C and H-J. H2O2 – Hydrogen Peroxide, GFP – Green fluorescent protein, ir – immunoreactivity, LGR – L-glutathione reduced, PINK1 – PTEN-induced putative kinase 1, TH – tyrosine hydroxylase.</p></div

The PINK1 promoter construct and expression pattern in zebrafish larva of the <i>Tg</i>(pink1:EGFP) line.

<div><p>A. Construction of the PINK1 promoter DNA fragment comprising the sequence -2 kb upstream of the 5’ end of the <i>pink1</i> gene and including the ATG start site. This is inserted in the cloning site in the Tol2 transposon vector pCS-cfosGFP, which contains a small intronic sequence and a GFP expression cassette having minimal cfos activity [21]. B-F. Confocal z-stack images of the whole-mount <i>Tg</i>(pink1:EGFP) larval stages.</p> <p>B. GFP expression as observed in peripheral tissues at different stages of development by GFP IHC. In a lateral view of 4 dpf whole mount larvae, expression was observed in the heart (H) and liver (L); anterior to the left. </p> <p>C. Expression in the muscle of the 4-dpf larvae in a lateral view (M); anterior to the left. </p> <p>D. The whole mount GFP expression at 5 dpf; dorsal views, anterior to the top, showing pronounced immunoreactive cells in brain regions such as the telencephalon (Tel), mid-hindbrain boundary (MHB) and rhombencephalon (rho).</p> <p>E- F. Dorsal and ventral views of the brain of 7-dpf <i>Tg</i>(pink1:EGFP) fish; anterior to the right. </p> <p>E. In the ventral view, the strongest GFP expression is seen in the telencephalon (Tel), anterior part of optic tectum (TeO), thalamus (T), and the lateral rhombencephalon (rho). F. The dorsal view shows the prominent expression in the mid-hindbrain boundary (MHB) along with Tel and rho.</p> <p>Scale bar represents 100μm. GFP – Green fluorescent protein, PINK1 – PTEN-induced putative kinase 1.</p></div

Expression levels of <i>th1</i> and <i>th2</i> mRNA at 3 dpf in <i>pink1</i> morphants untreated and treated with LGR.

<div><p>A-C. Altered <i>th1</i> expression in the untreated std ctrl MO, <i>pink1</i>MO, and <i>pink1</i>MO+mRNA groups. A decrease in <i>th1</i> expression in the population 5,6,11 (*) in morphants (B) was observed as compared to the ctrl MO group (A). The expression could be rescued by <i>pink1</i> mRNA co-injection (C). </p> <p>D. Representative quantitative graph for the untreated group.</p> <p>E-G. The expression of <i>th1</i> ISH in LGR-treated groups. The drug LGR rescued the reduced <i>th1</i> cells in the eyes and also group 5,6,11 of the morphants (E) as compared to the ctrl MO group (D). The expression is enhanced in the rescue group with the combined effect of LGR and <i>pink1</i> mRNA (F).</p> <p>H. Graphical representation of the transcript levels for <i>th1</i> in the LGR-treated group. A significant change in the <i>pink1</i> mRNA co-injected group.</p> <p>I-K. The expression of <i>th2</i> ISH in the untreated std MO, <i>pink1</i>MO, and <i>pink1</i>MO+mRNA groups. A loss of <i>th2</i> was observed in group 10b in the <i>pink1</i> morphants (H) as compared to ctrl MO (G). The <i>pink1</i> mRNA rescued <i>th2</i> expression in group 10b (I). </p> <p>L. The representative <i>th2</i> transcript levels determined by q-RT-PCR for the untreated group.</p> <p>M-O. The expression of <i>th2</i> ISH in LGR treated groups. LGR-treated <i>pink1</i> MO shows a slight increase in the <i>th2</i> 10b group (K) as compared to ctrl MO (J). The <i>pink1</i> mRNA along with LGR completely rescues the loss of <i>th2</i> in the 10b cell group (L). </p> <p>P. Representative q-RT-PCR results for the <i>th2</i> LGR-treated group. A significant change in the <i>pink1</i> mRNA co-injected group together with LGR significantly increased the <i>th2</i> transcript levels. </p> <p>Figures D and L present confirmation of <i>th1</i> and <i>th2</i> transcripts downregulation in the <i>pink1</i> morphants. Figures H and P present the additive rescue effect of LGR together with <i>pink1</i> mRNA on the morphants.</p> <p>The Bonferroni Hochberg multiple comparison statistical test was applied for the q-RT-PCR results (* p < 0.05, ** p < 0.01, *** p < 0.001). ISH – in-situ hybridization, LGR – L-glutathione reduced, MO – morpholino oligonucleotides, <i>pink1</i> – PTEN-induced putative kinase 1, q-RT-PCR – quantitative real-time PCR, <i>th1</i> – tyrosine hydroxylase1, <i>th2</i> – tyrosine hydroxylase 2.</p> <p>Scale bar represents 100 μm.</p></div

Loss of Wdfy3 in mice alters cerebral cortical neurogenesis reflecting aspects of the autism pathology.

Autism spectrum disorders (ASDs) are complex and heterogeneous developmental disabilities affecting an ever-increasing number of children worldwide. The diverse manifestations and complex, largely genetic aetiology of ASDs pose a major challenge to the identification of unifying neuropathological features. Here we describe the neurodevelopmental defects in mice that carry deleterious alleles of the Wdfy3 gene, recently recognized as causative in ASDs. Loss of Wdfy3 leads to a regionally enlarged cerebral cortex resembling early brain overgrowth described in many children on the autism spectrum. In addition, affected mouse mutants display migration defects of cortical projection neurons, a recognized cause of epilepsy, which is significantly comorbid with autism. Our analysis of affected mouse mutants defines an important role for Wdfy3 in regulating neural progenitor divisions and neural migration in the developing brain. Furthermore, Wdfy3 is essential for cerebral expansion and functional organization while its loss-of-function results in pathological changes characteristic of ASDs