1. Volume 95, Issue 4, Pages 834-851.e5 (August 2017)
Molecular Memory of Morphologies by Septins during Neuron Generation Allows Early Polarity Inheritance 
Leila Boubakar, Julien Falk, Hugo Ducuing, Karine Thoinet, Florie Reynaud, Edmund Derrington, Valérie Castellani 
Neuron 
Volume 95, Issue 4, Pages e5 (August 2017)
DOI: /j.neuron
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2. Figure 1
Morphological Differentiation of DRG Neurons and Process Inheritance during NCCs Division
(A and B) GFP expression reveals the morphology of NCCs populating DRGs in transverse sections of E4 embryos (A) (48 hr after electroporation) and E3 embryos (B) (24 hr after electroporation). 48 hr after electroporation, many GFP+ cells have extended long neurites (green arrowheads); in contrast, 24 hr after electroporation, the majority of GFP+ cells are still morphologically immature (red arrowheads).
(C) Pseudo-colored cell exemplifying the characteristic bipolar morphology of cells that had formed neurites.
(D) Histograms showing the percentage of cells that have two neurites (green) or more (red) in E3 and E4 embryos (n = 11 embryos).
(E) Typical examples of three different classes of morphologically immature cells were pseudo-colored from confocal stacks (bipolar, left; multipolar, middle; protrusive, right).
(F) Histograms showing the proportions of the three immature cell types in the DRG 24 hr after electroporation (n = 12 embryos).
(G) Morphology of GFP+ cells in the DRG from embryo slices at T0 (left) and after 9 hr (right) of recording. Note that the structure of the DRG is maintained and that cells formed neurites.
(H) Time-lapse sequence of a non-dividing NCC (green asterisk) undergoing morphological transition from protrusive to bipolar morphology. Arrowheads point to the neurites.
(I) Time-lapse sequence showing the morphological transition leading to bipolar neurons after cell division. Daughter cell processes formed at positions matching that of the progenitor processes.
(J) Time-lapse sequence illustrating process location inheritance after cell division. Right panel shows the overlay of cell outlines before (red) and after (green) cell division to emphasize the similarity between mother and daughter processes (green arrowheads).
(K) Neurofilament labeling of dissociated DRG neurons showing that they re-acquire their bipolar morphology in culture. Scale bars, 10 μm (A–C and E–J), 20 μm (K). Error bars represent SEM.
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3. Figure 2 Expression of Septins in DRG Neurons
(A) Model of axial budding in S. cerevisiae. An axial landmark (blue), which includes the Septins, tags the membrane of the mother cells (M) and controls the positioning of the bud (and future daughter cell, arrowhead).
(B) Schematic representation of the Septin subfamilies in vertebrates.
(C) RT-PCR showing the expression of the Septins 2, 3, 5, 7, and 11 in the spinal cord and the DRG of E4 embryos.
(D) Western blot analysis demonstrating—at protein levels—expression of Septins 2, 6, and 7 in the spinal cord and the DRG of E4 embryos.
(E) Immunodetection of Septin 7 in DRG dissociated cells after 1 hr of culture labels puncta and short filaments in the soma and the processes. Chevron-like structures are observed at the base of filopodia, as shown at higher magnification (right, arrowhead).
(F) Left: immature round neuron has a polarized localization of Septin 7 (green), forming a mark at the cell cortex (arrowhead). Nucleus (Islet labeling) is shown in red. Right: graphical representation of the mean fluorescence intensity of Septin 7 at the cell cortex of round DRG neurons (n = 33 cells). Scale bar, 10 μm (E and F).
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4. Figure 3
Septin 7 Distribution during NCC Division and Neuron Polarization
(A and B) Time-lapse sequences showing the dynamics of Septin7-GFP (Sept7-GFP) during division and process formation by daughter cells. Before division Sept7-GFP was present throughout progenitor soma and processes (A, T = −20 min; B, T = −10 min). During rounding for division, Sept7-GFP accumulates (A, yellow; B, green) at the two sites where the processes retracted, tagging their position in the soma (mitosis). Daughter cells rapidly form processes from these two accumulation sites (A and B, T = +10 min), and Sept7-GFP relocalizes at the opposite pole to the midbody.
(C and D) Quantified analysis showing the spatial correlation between Sept7-GFP accumulation during mitosis and process initiation site. Sept7-GFP intensity at the cell cortex was measured during mitosis (ROI section, left) (C). The site of process emergence was then visualized by measuring the RFP intensity, in the same ROI section, just after division (middle). The superposition of the two plot profiles shows the correlation between the two measurements (right). The central bar corresponds to process emergence, and the two gray bars correspond to the baseline (see STAR Methods). Histogram showing the specific accumulation of Sept7-GFP, during mitosis, at the site of process emergence (white bar, see also white selection in C, right) (D), compared to the area surrounding the process (black bar, see also gray selection in C, right) (n = 11 processes, p = 2.835E−6). Error bars represent SEM. ∗∗∗p <
(E) Details of the sequence presented in (C) showing the presence of Sept7-GFP structures (green, red arrow) at the base of the emerging protrusion (red).
(F and G) Time-lapse sequences showing the spatial correlation between Sept7-GFP accumulation and process formation during the polarization of DRG neurons in vitro. Note that, as the cell polarizes, Sept7-GFP (shown in fire LUT) progressively accumulates at the site of process emergence (green arrow). Profile plots of the normalized Sept7-GFP (% of maximum) are shown under the corresponding time points, and red lines delineate the site of protrusion (G). mRFP, membrane RFP.
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5. Figure 4 Septin Inhibition Impairs DRG Neuron Polarization
(A) Co-immunoprecipitations (IPs) from E4 spinal cord expressing Borg3WT-GFP (DN) showing the interaction between the DN construct and endogenous Septin 7 (middle) and Septin 2 (right).
(B) DN expression (green, top) and Septin 7 immunolabeling (gray, bottom) on transverse sections 24 hr after electroporation showing Septin 7 disorganization in both the spinal cord (left) and in the DRG (right).
(C) Septin 7 (gray) distribution alteration already seen 12 hr after electroporation of the DN construct (green).
(D) Photomicrographs of NCCs expressing control (left) and DN (right) constructs in the DRG 24 hr after electroporation. Note the absence of neurite bearing cells in the DN condition.
(E) Histograms showing the decrease of neurite bearing cells in DN+ cells compared to the control (Ctr.) (control: n = 18 embryos, 836 cells; DN: n = 23 embryos, 613 cells; p = 2.62E−7).
(F) Histograms showing morphological changes of immature cells between control and DN conditions (control: n = 12 embryos, 644 cells; DN: n = 17 embryos, 486 cells; p(bipolar) = 6.937E−6, p(multipolar) = 7.382E−5, p(protrusive) = ).
(G) Representative images of B103 cells, expressing Sept7-GFP construct, transfected with a control siRNA (top) or a siRNA directed against Sept7 (bottom). Note the decrease of Sept7-GFP intensity on the siRNA-treated condition.
(H) FACS analysis showing the efficiency of Sept7 knockdown in B103 cells 24 hr after transfection. Analysis was performed on the Q2 fraction of transfected (Tomato+) cells expressing the Sept7-GFP construct (triplicated experiments, p < 0.001).
(I) Sept7 immunolabeling showing the depletion of Sept7 in the spinal cord 24 hr after electroporation of the siRNA. Plot profiles represent the intensity of Sept7 labeling in the control (gray) and electroporated (red) sides of the spinal cord for the control (top) and the siRNA (bottom) conditions.
(J) Photomicrographs of NCCs expressing control (left) or siRNA (right) in the DRG 24 hr after electroporation.
(K) Histograms showing the decrease of neurite bearing cells in the siRNA+ population compared to the control one (Ctr.) (control: n = 4 embryos, 291 cells; siRNA #1: n = 4 embryos, 190 cells; p = ; siRNA #2: n = 4 embryos, 270 cells; p = ).
(L) Histograms showing morphological changes of immature cells between control and siRNA conditions (control: n = 4 embryos, 291 cells; siRNA #1: n = 4 embryos, 190 cells; p(bipolar) = , p(multipolar) = ; siRNA #2: n = 4 embryos, 270 cells; p(bipolar) = , p(multipolar) = ). Scale bars, 10 μm. Numbers above bars indicate the number of embryos. Error bars represent SEM, n.s. p > 0.05; ∗p < 0.05; ∗∗∗p <
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6. Figure 5
Septin Inhibition Blocks Neuronal Polarization, but Not Cell Survival or DRG Neuron Specification
(A) Photomicrographs of TUNEL labeling (red) on DRG sections containing NCCs expressing control (left) or DN (right) (green) constructs 24 hr after electroporation. Magnifications exemplify typical TUNEL labeling.
(B) Histograms showing the numbers of TUNEL+ cells in the spinal cord and DRG (control-spinal cord: n = 8 embryos; DN-spinal cord: n = 9 embryos; control-DRG: n = 8 embryos, DN-DRG: n = 8 embryos; p(spinal cord) = 0.88; p(DRG) = 0.12).
(C) Confocal images of Islet immunolabeling in DRG transfected with control (left) or DN (right) constructs. Islet staining was observed in GFP+ cells (green outlines) in both conditions.
(D) Histograms showing the percentage of GFP+/Islet+ cells between control (Ctr.) and the DN conditions (control: n = 10 embryos; DN: n = 14 embryos; p = ).
(E) Representative image of Islet immunostaining (red) in early bipolar neurons 24 hr after electroporation.
(F) Histograms of the percentage of neurite-bearing cells in control and DN conditions among Islet+ cells showing that the DN expression inhibits neurite formation (control: n = 10; DN: n = 14; p = ).
(G) Morphologically immature cells also express Islet (red). Islet is detected in bipolar (left) and protrusive (right) cells.
(H) Histograms showing the increase of non-bipolar morphologies in immature Islet+ cells expressing the DN (control: n = 10 embryos; DN: n = 14 embryos; p(bipolar) = , p(multipolar) = , p(protrusive) = ).
(I) Pseudo-colored photomicrograph of atypical multipolar shape observed in cells expressing the DN. Histograms (left) highlighting the appearance of this complex multipolar shape (more than five processes) in the DN condition. Scale bars, 10 μm (C, E, and G); 20 μm (A). Numbers above bars indicate the number of cells (B) or embryos (D, F, H, and I). Error bars represent SEM, n.s. p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <
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7. Figure 6
Septins Inhibition Impairs Morphological Transitions of Protrusive NCCs and Protrusion Inheritance after NCCs Division
(A) Images from time-lapse sequence showing lack of neurite formation in DRG cells expressing the DN construct in embryo slices.
(B) Monitoring of protrusive cells (red and white asterisks) showing that DN-expressing cells retain high protrusive activity as they persistently extend and retract protrusions (red and white arrowheads).
(C) Time-lapse sequences illustrating the spatial correlation found in control+, but not DN+, cells between the progenitor protrusion (T = −10 min before mitosis) and that formed by the daughter cells after mitosis (T = 10 and 20 or 30 min). Right panels show the overlay of cell outlines before and after division. Arrowheads point to the main protrusion.
(D) Histogram showing the loss of spatial correlation between the progenitor and daughter cell protrusions in DN+ cells (control: n = 51 protrusions; DN: n = 41 protrusions; p = 0.005).
(E) Monitoring of mitosis using H2BiRFP labeling in control+ (top) or DN+ (bottom) NCCs in the DRG. Beginning and end of mitosis are characterized by chromosome condensation and formation of two de-condensed nuclei, respectively.
(F) Dot plot showing the increase of cell mitosis duration in the DN condition (control: n = 99 cells; DN: n = 62 cells, p = ).
(G) Histogram showing the lack of correlation between mitosis duration and loss of spatial correlation between the progenitor and daughter cell protrusions in DN+ cells (control: n = 51 protrusions; DN N.: n = 20 protrusions; p = ; DN A.: n = 21 protrusions; p = ). N., normal mitosis duration (<30 min); A., altered mitosis duration (>30 min).
(H) Schematic representation and histogram showing the lack of correlation between division axis and site of emergence of protrusions in DN+ cells (control: n = 29 cells; DN: n = 12 cells; p = 0.003). Purple: protrusion emission is perpendicular to the division axis; blue: protrusion emission is non-correlated to the division axis. DN, fraction of DN+ cells with an impaired division axis. Scale bars, 10 μm (A, B, E, and H); 40 μm (C). Numbers indicate the number of protrusions (D and G) and cells (F and H). Error bars represent SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <
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8. Figure 7
Alteration of Septin Dynamics Impairs the Mechanism of Morphological Memory in DRG Neurons
(A and B) Inhibition of Septins by FCF treatment decreases the percentage of neurite-bearing DRG neurons in vitro. Phase images (A) of control and FCF-treated cultures and histograms (B) showing the percentage of neurite-bearing cells (i.e., having a process at least twice as long as their soma) (control: n = 9,073 cells; FCF: n = 9,579 cells).
(C) FCF treatment alters Septin 7 organization, leading to the formation of thick and swirled filaments.
(D) Schematic representation of the experimental procedure and the different expected outcomes. If Septin inhibition by FCF induces a loss of morphological memory, this should lead to an absence of process formation or to the acquisition of an abnormal, multipolar morphology. If the morphological memory is not altered by Septin inhibition, DRG neurons should be able to acquire their bipolar morphology.
(E) After recovery, DRG neurons are able to reform neurites (left histogram, FCF: n = 5, total 2,183 cells, Recovery: n = 6, total 2,586 cells Ctr.: n = 5, total 1,762 cells, FCF versus recovery: p = 0.004, Mann-Whitney), but the cells present an altered morphology, with an increase in the proportion of multipolar cells (right histogram, FCF: n = 5, total 135 cells, Recovery: n = 6, total 721 cells Ctr.: n = 5, total 469 cells, FCF versus recovery: p = 0.03; Ctr. versus recovery: p = 0.004; Mann-Whitney).
(F and G) In control condition and after Nocodazole removal, cells are able to reform their process at the exact same site as that of the retracted process. Cells treated with FCF fail to reform their processes at the same location as they were before Nocodazole treatment. Examples of time-lapse sequences in control (Ctr.) and FCF-treated conditions are shown in (F) and graphical representation of the initiation site inheritance is shown in (G) (DMSO versus FCF p = 7.23E−5, Fisher’s exact test, numbers refer to processes).
(H and I) Overexpression of Sept7-GFP alters the morphology of DRG neurons, leading to an increase of the percentage of multipolar cells. Illustration of morphologies of non-transfected and Sept7-GFP-expressing cells (H). Histograms showing the percentage of multipolar cells among non-transfected and Sept7-GFP cells (I), (n = 12, p = , Mann-Whitney, total number of GFP+ cells 626). Scale bars, 10 μm except 20 μm in (A). Error bars represent SEM.∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <
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