Active SadA and SadB are restricted to the axons of polarized neurons.

<p>(A) Coronal sections from the brains of E17 mouse embryos were stained with Hoechst 33342 (blue) an antibody detecting active SadA and SadB phosphorylated at Thr175 and Thr187, respectively, (P-SadA/B, green) and the Tuj1 (red) antibody. (B) Cortical neurons from E18 rat embryos were analyzed at 1, 2 and 3 days in vitro (d.i.v., stage 2 to 3) by staining Hoechst 33342 (blue) an antibody detecting active SadA and SadB phosphorylated at Thr175 and Thr187, respectively, (P-SadA/B, green) and the Tuj1 (red) antibody. Early and late stage 3 neurons are shown. Axons are marked by arrowheads. The scale bars are 20 μm (A) and 50 μm, respectively (B).</p

The loss of Sad kinases leads to a decrease in the number of neuronal progenitors in the cortex.

<p>(A-D) Coronal sections from the brains of E13, E15 or E17 embryos with the indicated genotypes were analyzed by staining with anti-Pax6 (APCs, A, pseudo-colored cyan), anti-Tbr2 (IPCs, B, green). Nuclei were stained with Hoechst 33342 (blue). The number of Pax6⁺ (B), Tbr2⁺ (D) cells per 10⁴ μm² area in ventricular surface was quantified in the <i>Sada</i>^<i>+/-</i><i>;Sadb</i>^<i>+</i>/- (magenta) and <i>Sada</i>^<i>-/-</i><i>;Sadb</i>^<i>-</i>/- cortex (green). Values are means ± s.e.m., n = 3 different embryos, two-way ANOVA compared to heterozygous control; *, p<0.05; **, p<0.01; ****, p<0.0001). The Scale bars are 50 μm.</p

The loss of the kinases SadA and SadB results in early neuronal apoptosis and a reduced number of progenitors

<div><p>The neurons that form the mammalian neocortex originate from progenitor cells in the ventricular (VZ) and subventricular zone (SVZ). Newborn neurons are multipolar but become bipolar during their migration from the germinal layers to the cortical plate (CP) by forming a leading process and an axon that extends in the intermediate zone (IZ). Once they settle in the CP, neurons assume a highly polarized morphology with a single axon and multiple dendrites. The AMPK-related kinases SadA and SadB are intrinsic factors that are essential for axon formation during neuronal development downstream of Lkb1. The knockout of both genes encoding Sad kinases (<i>Sada</i> and <i>Sadb</i>) results not only in a loss of axons but also a decrease in the size of the cortical plate. The defect in axon formation has been linked to a function of Sad kinases in the regulation of microtubule binding proteins. However, the causes for the reduced size of the cortical plate in the <i>Sada</i>^<i>-/-</i><i>;Sadb</i>^<i>-/-</i> knockout remain to be analyzed in detail. Here we show that neuronal cell death is increased and the number of neural progenitors is decreased in the <i>Sada</i>^<i>-/-</i><i>;Sadb</i>^<i>-/-</i> CP. The reduced number of progenitors is a non-cell autonomous defect since they do not express Sad kinases. These defects are restricted to the neocortex while the hippocampus remains unaffected.</p></div

Increased apoptosis in the <i>Sada</i>^<i>-/-</i><i>;Sadb</i>^<i>-/-</i> knockout brain.

<p>(A, C) Coronal sections from the cortex of E13, E14, E15 or E17 embryos with the indicated genotypes were stained with an anti-cleaved caspase-3 (A, green) or anti-phospho-Ser139-<b>γ</b>H2A.X antibody (B, red) and Hoechst 33342 (blue). (B, D) The number of nuclei per section positive for cleaved caspase-3 (B) or phospho-S139-<b>γ</b>H2A.X (D) was determined in the cortex at the indicated stages. No signals for phospho-Ser139-<b>γ</b>H2A.X were detectable in heterozygous controls. Values are means ± s.e.m., n = 3 different embryos, Student’s t-test n between E15 and E17 (*, p<0.05; **, p<0.01). The scale bars are 50 μm.</p

Defects in cortical development in the <i>Sada</i>^-/-;<i>Sadb</i>^-/- knockout.

<p>Defects in cortical development in the <i>Sada</i>^-/-;<i>Sadb</i>^-/- knockout.</p

The loss of Sad kinases leads to an increase in the mitotic index.

<p>(A, E) Coronal sections from the brains of E13, E15 or E17 embryos with the indicated genotypes were analyzed by staining with anti-Ki67 (A, proliferating cells, red) and anti-PH3 antibodies (A, cells in M-phase, green) or with an anti-PCNA antibody (E, green) and Hoechst 33342 (blue). (B—D) A significant decrease in the number of proliferating Ki67⁺ cells per 10⁴ μm² (B) was observed at E15 and E17 when comparing the <i>Sada</i>^<i>+/-</i><i>;Sadb</i>^<i>+</i>/- (magenta (B, C) and blue (D), respectively) and <i>Sada</i>^<i>-/-</i><i>;Sadb</i>^<i>-</i>/- cortex (green). The number of mitotic PH3⁺ cells per section remained constant (C) but the mitotic index (D, ratio of PH3⁺ and Ki67⁺cells in %) increased in the <i>Sada</i>^<i>-/-</i><i>;Sadb</i>^<i>-</i>/- cortex (green) at E15 and E17 compared to <i>Sada</i>^<i>+/-</i><i>;Sadb</i>^<i>+</i>/-controls since the number of Ki67⁺cells was reduced. (F—G) The number of PCNA⁺ cells in S-phase (PCNA⁺ cells with a punctate staining pattern (inset)) per 10⁴ μm² (F) and the proportion of cells in S-phase (G, ratio of PCNA⁺ S-phase and Ki67⁺cells in %) in the VZ/SVZ of the cortex were quantified in the <i>Sada</i>^<i>-/-</i><i>;Sadb</i>^<i>-</i>/- cortex (green) and <i>Sada</i>^<i>+/-</i><i>;Sadb</i>^<i>+</i>/-controls (magenta (F) and blue (G), respectively). Values are means ± s.e.m., n = 3 different embryos, two-way ANOVA compared to heterozygous control; *, p<0.05; **, p<0.01; ***, p<0.001, n. s., not significant). The Scale bars are 20 μm.</p

SadA and SadB can be detected in the CP and axons but not the VZ.

<p>(A) Coronal sections from the brains of E13, E15 or E17 mouse embryos were stained with Hoechst 33342 (blue) and an anti-SadA (green) antibody directed against the N-terminal kinase domain or an anti-Sad B antibody (green) directed against the C-terminus. (B) Cortical neurons from E18 rat embryos were analyzed at 1, 2 and 3 days in vitro (d.i.v., stage 2 to 3) by staining with anti-SadA (A, green), anti-SadB (A, green) and the Tuj1 (red) antibodies. Nuclei were stained with Hoechst 33342 (blue). Early and late stage 3 neurons are shown. Axons are marked by arrowheads. The scale bars are 20 μm (A) and 50 μm, respectively (B).</p

C3G/Rapgef1 Is Required in Multipolar Neurons for the Transition to a Bipolar Morphology during Cortical Development

<div><p>The establishment of a polarized morphology is essential for the development and function of neurons. During the development of the mammalian neocortex, neurons arise in the ventricular zone (VZ) from radial glia cells (RGCs) and leave the VZ to generate the cortical plate (CP). During their migration, newborn neurons first assume a multipolar morphology in the subventricular zone (SVZ) and lower intermediate zone (IZ). Subsequently, they undergo a multi-to-bipolar (MTB) transition to become bipolar in the upper IZ by developing a leading process and a trailing axon. The small GTPases Rap1A and Rap1B act as master regulators of neural cell polarity in the developing mouse neocortex. They are required for maintaining the polarity of RGCs and directing the MTB transition of multipolar neurons. Here we show that the Rap1 guanine nucleotide exchange factor (GEF) C3G (encoded by the <i>Rapgef1</i> gene) is a crucial regulator of the MTB transition <i>in vivo</i> by conditionally inactivating the <i>Rapgef1</i> gene in the developing mouse cortex at different time points during neuronal development. Inactivation of C3G results in defects in neuronal migration, axon formation and cortical lamination. Live cell imaging shows that C3G is required in cortical neurons for both the specification of an axon and the initiation of radial migration by forming a leading process.</p></div

C3G is required in multipolar neurons for neuronal polarization.

<p>(A-B) Wild type (WT) or <i>Rapgef1</i>^flox/flox (C3G f/f) E13.5 brains were transfected by <i>ex vivo</i> electroporation with (A) <i>pEF-Cre</i>, <i>pEF-LPL-LynN-EGFP</i> and <i>pTα-LPL-H2B-RFP</i> or (B) <i>pTα-Cre</i>, <i>pTα-LPL-LynN-EGFP</i> and <i>pTα-LPL-H2B-RFP</i> to specifically inactivate the conditional alleles and label early post-mitotic neurons. Imaging was performed 30h after electroporation. Neurons from WT coronal slices first extend a long trailing process followed by a leading process. Slices from <i>Rapgef1</i>^flox/flox brains showed a significant number of neurons that remained multipolar and did not extend a trailing or a leading process after more than 20 h of imaging. (C, D) The percentage of cells that formed only a trailing axon (unipolar (only axon), red), only a leading process (unipolar (only leading process), yellow), that became bipolar (blue), or remained multipolar (green) after transfection of <i>pEF-Cre</i> (C) or <i>pTα-Cre</i> (D) at the end of the imaging period of 20 h (means ± SEM, ***p ≤ 0.001, *p ≤ 0.05 two-way ANOVA with Tukey’s multiple comparison test; number of bipolar or multipolar neurons from <i>Rapgef1</i>^flox/flox slices compared to control slices; n = 37 (wildtype; EF), n = 29 (<i>Rapgef1</i>^flox/flox; EF) and n = 51 (wildtype; Tα), n = 45 (<i>Rapgef1</i>^flox/flox; Tα) from 3 independent experiments that each included multiple slices from different animals, n indicates the total number of neurons analyzed in all experiments). The VZ is to the bottom and the pial surface to the top. Scale bars are 20 μm.</p

Loss of active β1 integrin in the C3G^Emx1-KO cortex.

<p>(A,-C) Coronal sections from heterozygous or homozygous E15 C3G^Emx1-KO cortex were stained with an anti- integrin β1 (VLA, green) and an antibody specific for active form of β1 integrin (9EG7, green) (A). No significant differences were found in the expression of β1 integrin when intensity values were plotted for the control and the C3G^Emx1-KO. (B) The level of active β1 integrin was reduced at the pial surface in the C3G^Emx1-KO cortex (+/-: arrows, -/-: arrowheads). Intensity profiles of 9EG7 immunofluorescence signals (arbitrary units) measured from the VZ (bottom) to the pial surface (top) show a significant reduction in the intensity of 9EG7 signals at the pial surface in the C3G^Emx1-KO mutant cortex only in the MZ (in the last 40 pixel positions that include the glial endfeet) where active β1 integrins are enriched in control sections [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154174#pone.0154174.ref020" target="_blank">20</a>]. The fluorescence intensity values (arbitrary units) for staining with the VLA and 9EG7 antibodies were quantified at each pixel position along the ventricular to pial axis in a rectangular box comprising an area from the VZ to the MZ in sections from 3 different embryos per genotype. The significance of differences was calculated between means at each pixel position (means ± SEM, Student’s t-test to measure the difference in the means, *p ≤ 0.05; ns, not significant). (C) Higher magnification images of the pial surface stained with the above mentioned antibodies show defects in the 9EG7 staining in the C3G^Emx1-KO. Dorsal is to the top. Single confocal planes are shown. Scale bar is 50 μm (A, B) and 20 μm (C).</p