Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Characterization of the SOXB2 expression in the developing dorsal midbrain at E12.5. Coronal sections of SOX21 GFPn midbrains stained with BrdU (A) , MASH1 (B) , LHX2 and SOX14 (C) and GATA3 (D) antibodies. Coronal sections of SOX14 +/− midbrains were stained with BHLHB5 and SOX21 (E) , BHLHB5 and SOX2 (F) , SOX21 and GATA2 (G) , BHLHB5 and GATA2 (H) , BrdU and LHX2 (I) , GATA3 and LHX2 (K) and GAD6 (J) antibodies. The results of this analysis are summarized in the schematic (L) . Red, green and blue arrows indicate the corresponding single stained cells (as shown in the label) and yellow and purple arrows denote co-localization between green and red cells and blue and red, respectively. Scale bar, 150 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Expressing, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Analysis of lineage-determining factors in SOX21 −/− embryos at E12.5. The immunostaining of the lineage-determining factors MASH1 (A,A′) , GATA2 (B,B′) and DBX1 (C,C′) was similar between knockout ( ii ) and wild type embryos ( i ). A significant reduction in the BHLHB5 staining was observed in the Knockout embryos in comparison to wild type (D,D′) . All nuclei were counted dorsally to the ventrolateral midbrain sulcus (indicated by white arrow). These data were confirmed by RT-Q-PCR analysis of embryos at the same age (E) . Data are represented as mean ± SEM, n = 10 (5 pairs of mice). * n = 10 (5 pairs of mice), P = 0.0116, two-tailed Mann-Whitney test. ** n = 6 (3 pairs of mice), p = 0.025, DF = 1, Kruskal Wallis test. *** n = 6 (3 pairs of mice), p = 0.0253, DF = 1, Kruskal Wallis test. Scale bar, 150 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Immunostaining, Knock-Out, Staining, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Lentiviral knock down of SOX21, SOX14 and BHLHB5. GATA3 eGFP embryos were infected through intra amniotic injections at E8.5 with lentiviral shRNA vectors targeting BHLHB5 (PLKO.1-shBHLHB5; ii ), SOX21 (PLKO.1-shSOX21; iii ) and SOX14 (PLKO.1-shSOX14; iv ). The expression of GFP was reduced in the embryos infected with the SOX14 shRNA vector (A,E) . Immunostaining of the embryos with BHLHB5 showed a reduction when embryos were infected with PLKO.1-shBHLHB5 and PLKO.1-shSOX21 (B) . Not surprisingly, SOX21 was only reduced when embryos were infected with PLKO.1-shSOX21 (C) while not noticeable changes were observed in the expression of SOX2 (D) and LHX2 (E) when infected with either vector. These data were confirmed by RT-Q-PCR analysis of embryos at E12.5 (F) . Data are represented as mean ± SEM. * n = 6 (3 pairs of mice), SOX21sh p = 0.0417, DF = 4, Kruskal Wallis test, ** n = 6 (3 pairs of mice), SOX14sh p = 0.0141, DF = 4, Kruskal Wallis test, *** n = 6 (3 pairs of mice), SOX21sh p = 0.016, BHLHB5sh p = 0.0474, DF = 4, Kruskal Wallis test, **** n = 6 (3 pairs of mice), SOX21sh p = 0.0064, SOX14sh p = 0.0021, DF = 4, Kruskal Wallis test. Scale bar, 150 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Knockdown, Infection, shRNA, Expressing, Plasmid Preparation, Immunostaining

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Overexpression of SOX21, SOX14 and BHLHB5 through in utero electroporation. Wild type embryos ( i,ii ) were in utero electroporated at E11 with Plasmids encoding CMV-SOX21-FLAG-IRES-GFP n , CMV-SOX14-FLAG-IRES-GFP n and CMV-BHLHB5-IRES-GFP n . The immunostaining of the embryos with BHLHB5 illustrated that ectopic expression of this gene could be achieved when SOX21 was overexpressed (A) and not with SOX14 (C) . Expression of GATA3 on the other hand was only induced when SOX14 was overexpressed (D) and not with SOX21 (B) . These experiments were repeated on SOX21 −/− embryos ( iii,iv ) providing similar findings (A’,B’,C’,D′) . Interestingly, ectopic expression of BHLHB5 was unable to induce either the GABAergic or Glutamatergic lineage as show by staining of the embryos with GATA3 or LHX2 (E,F) . Scale bar, 75 μm.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Over Expression, In Utero, Electroporation, Immunostaining, Expressing, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Sequential Role of SOXB2 Factors in GABAergic Neuron Specification of the Dorsal Midbrain

doi: 10.3389/fnmol.2018.00152

Figure Lengend Snippet: Schematic representation of the SOX2B during maturation of GABAergic neurons. SOX21 is expressed both in radial glial and neuronal progenitors at the VZ while been maintained in neuronal precursors at the sub ventricular zone (SVZ) where it appears to govern the expression of BHLHB5. At the MZ SOX14 marks the GABAergic lineage while promoting GATA3 expression.

Article Snippet: Lentiviral CMV-GFP (pLenti CMV GFP Puro) and shRNA vectors targeting SOX21 (PLKO.1-shSOX21), SOX14 (PLKO.1-shSOX14) and BHLHB5 (PLKO.1-shBHLHB5) were constructed according to the protocol of PLKO.1-puro vector (Addgene, USA) using the oligos in Supplementary Table S1.

Techniques: Expressing