Nelson et al. (2013): Dynamic interactions between Intermediate Neurogenic Progenitors and Radial Glia in embryonic mouse neocortex: potential role in Dll1-Notch signaling

 

  INPvz apical process filopodia and basal process dynamics. High-resolution live-cell multiphoton microscopy (MPM) was used to visualize INP cell dynamics in living E12.5 Tbr2GFP neocortical brain slices. INPvz exhibit different morphological states and exhibit both apical (tangential process extension, blue arrow/arrowhead) and basal process dynamics (pink arrow/arrowhead). INPsvz apical and INPvz basal processes interact (pink arrow/arrowhead), forming a transient type of radially oriented cell-contact. Time: 3h:20m (out of 7h:30m), 10min interval, 3 frames/sec playback.
Figure 1C: INPsvz apical process branching. High-resolution live-cell multiphoton microscopy (MPM) was used to visualize INP cell dynamics in living E14.5 Tbr2GFP neocortical brain slices. Similar Tbr2GFP + INP dynamics are observed in E14.5 acute neocortical slices. Note the highly branched processes and dynamics of the INPsvz (blue asterisk/arrows. Time: 2h:20m, 10min interval, 3 frames/sec playback.
Figure 1D: INPsvz apical process dynamics. High-resolution live-cell multiphoton microscopy (MPM) was used to visualize INP cell dynamics in living E14.5 Tbr2GFP neocortical brain slices. Note the extensive morphological changes in some INPsvz apical processes near the ventricular surface (pink and blue arrows). Time: 2h:20m, 10min interval, 3 frames/sec playback.
Figure 2D: RGvz hook morphology. Constitutively active membrane-targeted RFP (CAGGS-membrane-tdTomato, mTom) was electroporated ex utero into E14.5 Tbr2GFP neocortex, slices were cultured overnight, and analyzed by high-resolution 2-color live-cell MPM imaging the following morning. High-power view of digitally isolated RGvz (mTom+/Tbr2GFP-, red channel only, white asterisk) reveals more extensive membrane contact during hook-like formation in the apical process (arrows/arrowhead) surrounding an adjacent unlabeled RGvz division (mTom-/Tbr2GFP-, blue asterisk): note the dynamic tangential filopodia-like processes emanating from an upper region of the apical process (boxed region). Time: 3h:8m, 10min interval, 3 frames/sec playback.
Figure 2F: RGvz tangential processes. Constitutively active membrane-targeted RFP (CAGGS-membrane-tdTomato, mTom) was electroporated ex utero into E14.5 Tbr2GFP neocortex, slices were cultured overnight, and analyzed by high-resolution 2-color live-cell MPM imaging the following morning. High-power view in the VZ of an isolated RGvz/INPvz/INPsvz interaction reveals extensive contact between the cells, as well as a transient long-range dynamic filopodia (lower right, small blue arrows, dashed region) extending tangentially from the apical RGvz process (white asterisk/arrows). Also note the multiple tangential processes from the INPsvz (upper right, small blue arrows, dashed region) and the INPvz (lower left, dashed region).Time: 3h:38m, 10min interval, 3 frames/sec playback.
Figure 2G: INPvz tangential processes. Constitutively active membrane-targeted RFP (CAGGS-membrane-tdTomato, mTom) was electroporated ex utero into E14.5 Tbr2GFP neocortex, slices were cultured overnight, and analyzed by high-resolution 2-color live-cell MPM imaging the following morning. High-power view in the VZ of an isolated INPvz reveals a long-range dynamic filopodia (small blue arrow) extending tangentially from the apical process (bottom white arrow). Also note the multiple dynamic fine processes emanating from the soma/apical process (blue dashed region), in addition to the thicker dynamic short basal process (top white arrow).Time: 3h:38m, 10min interval, 3 frames/sec playback.
RGvz apical endfoot dynamics. Constitutively active membrane-targeted RFP (CAGGS-membrane-tdTomato, mTom) was electroporated ex utero into E14.5 Tbr2GFP neocortex, slices were cultured overnight, and analyzed by high-resolution 2-color live-cell MPM imaging the following morning. High-power view in the VZ of an isolated RGvz apical endfoot (blue arrow reveals extensive dynamic processes extending into the periventricular region (between 1-10 over this time period). Time: 12h:22m, 10 min interval, 3 frames/sec playback.
Figure 3C: Radial Relay 1. Constitutively active membrane-targeted RFP (CAGGS-membrane-tdTomato, mTom) was electroporated ex utero into E14.5 Tbr2GFP neocortex, slices were cultured overnight, and analyzed by high-resolution 2-color live-cell MPM imaging the following morning. High-power view of the SVZ/VZ reveals INPsvz cells (mTom+/Tbr2GFP+) form clusters in the SVZ, making radially oriented cell contacts with more apically located INPsvz that in turn, make additional radial contacts via its dynamic apical process with underlying INPvz (mTom-/Tbr2GFP+) and presumptive RGosvz (mTom+/Tbr2GFP-, white asterisk) (blue curved dashed arrows), which subsequently withdraws its apical attachment and undergoes basal somal translocation and mitosis (two white arrows) in the basal region of the VZ. Time: 3h:38m, 10min interval, 3 frames/sec playback.
Figure 3D: Radial Relay 2. Constitutively active soluble RFP (CMV-tdTomato, tdTom) was electroporated ex utero into E14.5 Tbr2GFP neocortex, slices were cultured overnight, and analyzed by high-resolution 2-color live-cell MPM imaging the following morning. High-power view of the SVZ/VZ reveals frequent and extensive dynamic radial relays of cell-cell interactions from INPsvz and RGosvz to underlying INPvz and RGvz in the basal niche. This movie (red channel only, gray scaled) is from the detailed time series in Figure 3D. Two well-defined pairs of radial relays are seen in this movie (left blue dash, right pink dash). On the right, radial relay contacts are seen from basally located INPsvz to more apically located INPsvz to newly divided RGvz (pink asterisks, curved arrows, respectively). On the left, radial relay contacts are seen from basally located INPsvz to more apically located RGosvz and RGvz (blue asterisks, curved arrows, respectively): note the RGosvz mitosis (small blue arrows). Time: 3h:50m, 10min interval, 3 frames/sec playback.
Figure 3F: RGosvz interactions with RGvz. Constitutively active membrane-targeted RFP (CAGGS-membrane-tdTomato, mTom) was electroporated ex utero into E14.5 Tbr2GFP neocortex, slices were cultured overnight, and analyzed by high-resolution 2-color live-cell MPM imaging the following morning (green channel not shown). Live-imaging of interactions between RGosvz (left cell, blue arrow/arrowheads) and the basal process of neighboring RGvz (dashed regions) reveals extensive RGosvz apical process dynamics, as well as shorter transient tangential filopodial-like protrusions from its basal process, both of which contact the neighboring RGvz basal process and induce reciprocal transient protrusions. Time: 3h:38m, 10 min interval, 3 frames/sec playback.
Figure 8 A1: Diverse Dll1-active progenitors. Ex utero electroporation of the destabilized mouse Dll1d2YFP reporter construct into wildtype E12.5 neocortices, and cultured 1DIV, and then analyzed by high-resolution live-cell MPM revealed Dll1 activity within all neocortical progenitor subtypes, all of which extended/retracted dynamic radial, multipolar, and/or tangential processes. Dll1-active INPsvz project apically directed filopodia (pink arrow/arrowheads) that contact underlying mitotic INPsvz (yellow arrows), which then also begin to project apically directed filopodia into the VZ post-division. Also note the Dll1-active INPvz with dynamic basal process (blue arrowhead) and apical process (blue arrow), which maintains contact with a apically dividing Dll1+ progenitor after the through the hook-phase. Time: 5h:36m, 12 min interval, 3 frames/sec playback.
Figure 8 B: Dll1-active progenitors extend tangential filopodia. Ex utero electroporation of the destabilized mouse Dll1d2YFP reporter construct into wildtype E12.5 neocortices, and cultured 1DIV, and then analyzed by high-resolution live-cell MPM reveals an isolated clonal pair of Dll1-active RGvz (blue arrow/arrowhead) and INPvz (pink arrow/arrowhead) extend dynamic tangential processes from their apical attachments and/or processes. Time: 5h:36m, 12 min interval, 3 frames/sec playback.
Figure 8D: Dll1-active INPsvz. Ex utero electroporation of the destabilized mouse Dll1d2YFP reporter construct into wildtype E14.5 neocortices, and cultured 2DIV, and then analyzed by high-resolution live-cell MPM reveals an isolated Dll1-active INPsvz undergoing morphological changes from multipolar to repeated apical process extensions and retractions. Time: 2h, 12 min interval, 3 frames/sec playback.
Figure 8 F3: Dll1-active INPsvz/INPvz radial interactions 1. Ex utero electroporation of the destabilized mouse Dll1d2YFP reporter construct into wildtype E14.5 neocortices, and cultured 1DIV, and then analyzed by high-resolution live-cell MPM reveals an isolated Dll1-active INPsvz/INPvz pair. Note the radial contact between the INPsvz (pink arrow) and INPvz (blue arrowhead), which leads to the apical translocation and mitosis at the VZ surface (blue arrow/arrowheads) for the INPvz after the INPsvz retracts its long apical process (pink arrowhead). Also note the transient hook-like displacement of the INPvz apical process due to an unlabeled neighboring progenitor division (yellow asterisk). Time: 9h:48m, 12 min interval, 3 frames/sec playback.
Figure 8G: Dll1-active INPsvz/INPvz radial interactions 2. Ex utero electroporation of the destabilized mouse Dll1d2YFP reporter construct into wildtype E14.5 neocortices, and cultured 1DIV, and then analyzed by high-resolution live-cell MPM reveals an isolated Dll1-active INPsvz/INPvz pair. Note the radial contact between the INPsvz (yellow arrow) and the INPvz (blue arrowhead), which in this case leads to the retraction of the INPvz process (blue arrows) and its basal translocation and mitosis (blue arrowheads), after the INPsvz retracts its apical process and migrates basally. Note the transient hook-like displacement of the INPvz apical process (blue arrows) due to an unlabeled neighboring progenitor division (white asterisk). Also note the neighboring Dll1-active INPvz that undergoes apical translocation and divides at the ventricular surface (pink arrow/arrowheads). Time: 7h:24m, 12 min interval, 3 frames/sec playback.
Figure 8H: Dll1-active apical and basal divisions. Ex utero electroporation of the destabilized mouse Dll1d2YFP reporter construct into wildtype E14.5 neocortices, and cultured 1DIV, and then analyzed by high-resolution live-cell MPM reveals Dll1 is active in both basal (pink arrows/arrowheads) and symmetric apical dividing progenitors (cleavage plane angle = 90o): also note that the apical process and membrane is split and equally inherited in each daughter (blue arrows). Time: 4h:48m, 12 min interval, 3 frames/sec playback.
Figure 8I: Dll1-active apical progenitor tangential interactions. Ex utero electroporation of the destabilized mouse Dll1d2YFP reporter construct into wildtype E14.5 neocortices, and cultured 1DIV, and then analyzed by high-resolution live-cell MPM reveals apically dividing Dll1-active progenitors can extend tangential dynamic filopodia right after dividing (blue arrowheads). Time: 5h:24m, 12 min interval, 3 frames/sec playback.

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