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. 2018 Apr 26;13(4):e0196698.
doi: 10.1371/journal.pone.0196698. eCollection 2018.

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

Pratibha Dhumale  1   2 , Sindhu Menon  1 , Joanna Chiang  1   2 , Andreas W Püschel  1   2
Affiliations

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

Pratibha Dhumale et al. PLoS One. .

Abstract

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 (Sada and Sadb) 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 Sada-/-;Sadb-/- 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 Sada-/-;Sadb-/- 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Reduced cortical size and loss of axons in Sada-/-;Sadb-/- embryos.
Coronal sections from the brains of Sada+/-;Sadb+/- and Sada-/-;Sadb-/- E17 embryos were analyzed by (A) Hematoxylin-Eosin staining or (B) staining with Hoechst 33342 (blue) and an anti-NF-M antibody (2H3) to mark the axons in the IZ of the cortex and the hippocampus. The boundaries between VZ/SVZ, IZ and CP are indicated by broken lines. The thalamocortical projection (marked by an asterisks) is missing in the Sada-/-;Sadb-/- cortex. The scale bars are 100 μm (A, B) and 50 μm (insets, B), respectively.
Fig 2
Fig 2. A loss of neurons in the Sada-/-;Sadb-/- cortex is first observed at E15.
(A, C, E) Coronal sections from the brains of E13 to E17 embryos with the indicated genotype were stained with antibodies for Tbr1+ (marker for L5/6 and subplate; green) or Ctip2+ (L4–6; red) and Hoechst 33342 (blue). (B, D, F) The number of Tbr1+ (B, F) and Ctip2+ cells (D, F) in the cortex (B, D) and hippocampus (F, E17) normalized to an area of 104 μm2 is shown for the Sada+/-;Sadb+/- (magenta) and Sada-/-;Sadb-/- 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<0.001. The scale bars are 50 μm.
Fig 3
Fig 3. The loss of Sad kinases leads to a decrease in the number of neuronal progenitors in the cortex.
(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 Sada+/-;Sadb+/- (magenta) and Sada-/-;Sadb-/- 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.
Fig 4
Fig 4. The loss of Sad kinases leads to an increase in the mitotic index.
(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 Sada+/-;Sadb+/- (magenta (B, C) and blue (D), respectively) and Sada-/-;Sadb-/- 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 Sada-/-;Sadb-/- cortex (green) at E15 and E17 compared to Sada+/-;Sadb+/-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 Sada-/-;Sadb-/- cortex (green) and Sada+/-;Sadb+/-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.
Fig 5
Fig 5. SadA and SadB can be detected in the CP and axons but not the VZ.
(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).
Fig 6
Fig 6. Active SadA and SadB are restricted to the axons of polarized neurons.
(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).
Fig 7
Fig 7. Increased apoptosis in the Sada-/-;Sadb-/- knockout brain.
(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-γH2A.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-γH2A.X (D) was determined in the cortex at the indicated stages. No signals for phospho-Ser139-γH2A.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.
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References

    1. Florio M., and Huttner W.B. (2014). Neural progenitors, neurogenesis and the evolution of the neocortex. Development 141, 2182–2194. doi: 10.1242/dev.090571 - DOI - PubMed
    1. Noctor S.C., Martinez-Cerdeno V., Ivic L., and Kriegstein A.R. (2004). Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nat Neurosci 7, 136–144. doi: 10.1038/nn1172 - DOI - PubMed
    1. Sakakibara A., and Hatanaka Y. (2015). Neuronal polarization in the developing cerebral cortex. Front Neurosci 9, 116 doi: 10.3389/fnins.2015.00116 - DOI - PMC - PubMed
    1. Funahashi Y., Namba T., Nakamuta S., and Kaibuchi K. (2014). Neuronal polarization in vivo: Growing in a complex environment. Curr Opin Neurobiol 27, 215–223. doi: 10.1016/j.conb.2014.04.009 - DOI - PubMed
    1. Namba T., Funahashi Y., Nakamuta S., Xu C., Takano T., and Kaibuchi K. (2015). Extracellular and Intracellular Signaling for Neuronal Polarity. Physiol Rev 95, 995–1024. doi: 10.1152/physrev.00025.2014 - DOI - PubMed

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This work was supported by the Deutsche Forschungsgemeinschaft (http://www.dfg.de) PU-102-12 (A.W.P.) and EXC 1003 - CiM (A.W.P.). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.