Severe NDE1-mediated microcephaly results from neural progenitor cell cycle arrests at multiple specific stages

Abstract

Microcephaly is a cortical malformation disorder characterized by an abnormally small brain. Recent studies have revealed severe cases of microcephaly resulting from human mutations in the NDE1 gene, which is involved in the regulation of cytoplasmic dynein. Here using in utero electroporation of NDE1 short hairpin RNA (shRNA) in embryonic rat brains, we observe cell cycle arrest of proliferating neural progenitors at three distinct stages: during apical interkinetic nuclear migration, at the G2-to-M transition and in regulation of primary cilia at the G1-to-S transition. RNAi against the NDE1 paralogue NDEL1 has no such effects. However, NDEL1 overexpression can functionally compensate for NDE1, except at the G2-to-M transition, revealing a unique NDE1 role. In contrast, NDE1 and NDEL1 RNAi have comparable effects on postmitotic neuronal migration. These results reveal that the severity of NDE1-associated microcephaly results not from defects in mitosis, but rather the inability of neural progenitors to ever reach this stage.

Figure 1. Effects of NDE1 and NDEL1 RNAi on neuronal migration into the cortical plate.

(a) Representative images of embryonic day 20 (E20) rat neocortex with a control vector expressing GFP alone, or shRNAs to NDE1, NDEL1, or both genes along with a GFP reporter. Sections were stained for Tbr1 to mark neurons in the cortical plate (CP). Scale bar, 50 μm. (b) Quantification of the fraction of electroporated cells in the CP across NDE1, NDEL1 or combined RNAi conditions 4 days post electroporation at E16. All knockdown conditions nearly eliminated any cells from reaching the CP, in comparison with control neurons. Data are presented as mean±s.e.m., unpaired t-tests used for all comparisons. *P<0.05, n=3 embryonic brains from different mothers.

Figure 2. NDE1 knockdown blocks apical nuclear migration and potently reduces the mitotic index.

(a) Live-imaging montage of GFP-expressing RGP cells at E19 with a control empty vector expressing GFP alone, or shRNAs to NDE1, NDEL1 or both genes along with a GFP reporter. Representative tracings from multiple RGP cells for each condition are shown at right. Montage panels are shown at 30 min intervals (Supplementary Movies 1–4). (b) Representative images of the VZ from the electroporated brains stained for the mitotic marker phosphohistone-H3 (PH3). Arrowheads mark soma of PH3+/GFP+ RGP cells. Dashed line represents the ventricular surface. (c,d) Measurements of the distance between the bottom of the nucleus and the ventricular surface, corresponding to the apical process length, across the various conditions. NDE1 knockdown shifted the apical process length distribution towards shorter distances, with a significant accumulation of RGPs with an apical process of 0–15 μm. NDE1/NDEL1 double knockdown, however, shifted the apical process length distribution to larger distances, with a significant accumulation of RGPs with an apical process of 30–45 μm. Each dot represents an individual apical process length measurement for one electroporated RGP cell. (e) Effect of RNAi on RGP cell mitotic index, measured as the number of electroporated RGP cells positive for PH3 divided by the total number of electroporated RGP cells. All mitotic figures of RGP cells were located at the ventricular surface, and NDE1 knockdown, as well as NDE1/NDEL1 double knockdown, caused a strong reduction in the mitotic index. Data presented as scatterplot in c with bars representing the median±the interquartile range, and as mean±s.e.m. in d and e. Kolmogorov–Smirnov test for non-parametric distributions used in c (*P<0.05, n=1,012–1,073 RGP cells). Unpaired t-test used in d and e (*P<0.05, n=3 embryonic brains from different mothers). Scale bar, 10 μm.

Figure 3. Double knockdown of NDE1/NDEL1 arrests cells at the G1-to-S transition.

E16 rat embryonic brains were electroporated with shRNAs for the various conditions and examined at E19. CyclinD1 staining was used to mark G1 cells, a 30 min pulse of BrdU used to mark S-phase cells, and BrdU pulses of varying length were used to distinguish RGPs that are arrested during the cell cycle from those actively cycling. The CyclinD1 and BrdU indices used in quantification were calculated as the amount of electroporated radial glia progenitors (RGPs) positive for either marker divided by the total number of electroporated RGP cells. (a) NDE1 knockdown caused a small but significant increase in CyclinD1-positive RGP cells, and the NDE1/NDEL1 double knockdown caused an even more substantial doubling of CyclinD1-positive RGP cells. There was no apparent difference between NDEL1 knockdown and control conditions. CylinD1-positive RGP cells tended to have soma located further away from the ventricular surface. Arrowheads mark electroporated RGP nuclei positive for CyclinD1. (b) NDE1 and NDE1/NDEL1 double knockdown caused a reciprocal and severe decrease in BrdU-labelled RGP cells. Again there was no significant difference between NDEL1 knockdown and control conditions. Arrowheads mark electroporated RGP nuclei positive for BrdU. (c) BrdU pulses of varying length revealed an increase in BrdU incorporation among control RGPs and NDEL1 knockdown RGPs, in contrast to the very minimal increase in BrdU incorporation over 24 h in the NDE1 and NDE1/NDEL1 knockdown conditions. Unpaired t-tests comparing knockdown conditions at each hour revealed no significant difference between control and NDEL1 knockdown, or NDE1 and NDE1/NDEL1 knockdown, while those two pairings were significantly different at every time point observed. Asterisks mark electroporated RGP cells positive for BrdU. Data are presented as mean±s.e.m. Unpaired t-tests used to compare conditions, *P<0.05, n=3 embryonic brains from different mothers. Scale bar, 10 μm in a–c. See Supplementary Figs 2 and 3 for further information regarding proliferation status cell cycle of knockdown RGP cells. Dashed line indicates ventricle surface.

Figure 4. The G1-to-S arrest of radial glia progenitors in the NDE1/NDEL1 double knockdowns disrupts the regulation of primary cilia length.

E16 rat embryonic brains were electroporated with the ciliary membrane marker Arl13B and shRNAs to the various conditions described below. All analyses were done at E19. (a,b) NDE1 knockdown resulted in a significant increase in primary cilia length among electroporated radial glia progenitors (RGPs), though the NDE1/NDEL1 knockdown caused an even greater doubling of primary cilia length. NDEL1 knockdown resulted in no change from control RGP cilia length. (c–e) Inhibition of primary cilia assembly by knockdown of the intraflagellar transport protein IFT172 rescued the CyclinD1 accumulation seen in the NDE1/NDEL1 double knockdown (e). The distribution of apical process lengths of the NDE1/NDEL1/IFT172 triple knockdown more closely mirrored the NDE1 knockdown alone (Fig. 2c). Arrowheads mark electroporated RGP cells positive for CyclinD1. Dashed line indicates ventricle surface. Data are presented as mean±s.e.m. Unpaired t-tests used to compare conditions in b and e, Kolmogorov–Smirnov test used for non-parametric distributions in d, *P<0.05, n=3 embryonic brains from different mothers. Scale bar, 10 μm in a and c, and 2.5 μm in insert panels for a. Also see Supplementary Figs 4 and 5.

Figure 5. NDE1 or NDEL1 overexpression largely rescues the neuronal migration defects seen after knockdown of either protein.

To test for RNAi rescue and functional complementation, embryonic rat brains were co-electroporated at E16 with shRNA to the various conditions with cDNA encoding RNAi-resistant proteins for self-rescue, and standard cDNA for cross-rescue. All the analyses were done at E20. Quantification of the amount of electroporated cells migrating into the cortical plate (CP) is shown at right. (a) Overexpression of NDE1 and NDEL1 produced no significant change in the amount of neurons migrating into the CP. (b) NDE1 knockdown with overexpression of RNAi-resistant NDE1 rescued neuronal migration into the CP. Overexpression of NDEL1 during NDE1 knockdown partially rescued neuronal migration into the CP. (c) Overexpression of RNAi-resistant NDEL1 or NDE1 with NDEL1 knockdown rescued the neuronal migration into the CP, even increasing the fraction of electroporated cells in the CP. Data are presented as mean±s.e.m., and unpaired t-tests used for all comparisons, *P<0.05, n=3 embryonic brains from different mothers. Scale bar, 50 μm.

Figure 6. RNAi-resistant NDE1 overexpression rescues all defects seen in radial glia progenitors across knockdown conditions.

RNAi-resistant NDE1 was co-electroporated into embryonic rat brains with a GFP control empty vector or along with NDE1 shRNA or NDE1 and NDEL1 shRNAs at E16, and analysed at E20. (a) Restoration of apical interkinetic nuclear migration, mitosis and subsequent basal migration of progeny measured by live imaging. Arrowheads marks the radial glia progenitor (RGP) of interest. Montage panels are shown at 30 min intervals. Full movie can be found in Supplementary Material (see Supplementary Movie 5), as well as an additional movie that more clearly displays the two-daughter cell progeny (Supplementary Movie 6). (b) Representative images of RGPs stained for PH3 within the VZ in various specified co-expression conditions. Arrowheads mark mitotic electroporated RGPs. Dashed line indicates ventricular surface. (c) Soma position of RGPs with RNAi-resistant NDE1 overexpressed during NDE1 knockdown indicates that the somal positioning distribution is rescued. (d) Overexpression of RNAi-resistant NDE1 with NDE1 knockdown also rescues the mitotic index. (e) Representative image of NDE1/NDEL1 double knockdown with overexpression of RNAi-NDE1, stained for PH3. Arrowheads mark mitotic electroporated RGPs. Dashed line indicates the ventricular surface. (f,g) Overexpression of RNAi-resistant NDE1 with double NDE1/NDEL1 knockdown rescues the distribution of RGP nuclei in the VZ and restores the mitotic index of RGP cells. Data are presented as scatterplot in c and f with bars representing the median±the interquartile range, and as mean±s.e.m. in d and g). Kolmogorov–Smirnov test for non-parametric distributions used in c and f (*P<0.05, n=428–474 RGP cells in c and n=224–260 RGP cells in f). Unpaired t-test used in d and g (*P<0.05, n=3 embryonic brains from different mothers). Scale bars, 10 μm. Also see Supplementary Fig. 6.

Figure 7. NDEL1 overexpression rescues apical nuclear migration but not mitotic entry in NDE1 deficient radial glia progenitors.

cDNA for NDEL1 was co-electroporated into embryonic rat brains with a GFP control empty vector or along with NDE1 shRNA or NDE1 and NDEL1 shRNAs at E16, and analysed at E20. (a) NDEL1 overexpression rescues apical interkinetic nuclear migration (INM) in radial glia progenitors (RGPs) where NDE1 has been knocked down, but these cells accumulate at the ventricular surface after apical INM, where they remain for hours without any evidence of mitosis. Montage panels are shown at 30 min intervals. Full movie can be found in Supplementary Movie 7). (b) Representative images of NDEL1 overexpression on both a wild-type and NDE1 RNAi background reveals an accumulation of the majority of RGP nuclei at the ventricular surface in a PH3 negative state. Arrowheads mark electroporated cells in mitosis. Dashed line represents the ventricular surface. (c) NDEL1 overexpression causes nearly all RGP soma to accumulate at the ventricular surface regardless of whether NDEL1 is overexpressed on a wild-type or NDE1 RNAi background. (d) Even though NDEL1 overexpression caused an accumulation of RGP soma at the ventricular surface, the mitotic index remained reduced to a level similar to NDE1 knockdown alone. (e) Representative image of RNAi-resistant NDEL1 overexpression with NDE1/NDEL1 double knockdown in RGP cells stained for PH3. Dashed line represents the ventricular surface. (f,g) RNAi-resistant NDEL1 overexpression with NDE1/NDEL1 double knockdown caused an accumulation of RGP soma at the ventricular surface similar to overexpression of NDEL1 on a wild-type or NDE1-deficient background, and once again failed to rescue the mitotic index. Data are presented as scatterplot in c and f with bars representing the median±the interquartile range, and as mean±s.e.m. in d and g. Kolmogorov–Smirnov test for non-parametric distributions used in c and f (*P<0.05, n=405-475 RGP cells in c and n=245–272 RGP cells in f). Unpaired t-test used in d and g (*P<0.05, n=3 embryonic brains from different mothers). Scale bars, 10 μm. Also see Supplementary Fig. 7.

Figure 8. BicD2 overexpression rescues apical nuclear migration but not entry into mitosis in radial glia progenitors depleted of NDE1.

cDNA for full-length BicD2 was co-electroporated into embryonic rat brains with a GFP control empty vector or along with NDE1 shRNA or NDE1 and NDEL1 shRNAs at E16, and analysed at E20. (a) The overexpression of BicD2 in radial glia progenitors (RGPs) lacking NDE1 restores apical migration, though the soma accumulate at the ventricle for hours without any evidence of mitosis. Montage panels are shown at 30 min intervals. Full movie can be found in Supplementary Movie 8. (b) Representative images of BicD2 overexpression on both a wild-type and NDE1 knockdown background with staining for PH3. Arrowheads mark mitoses in electroporated cells. Dashed line indicates ventricular surface. (c) BicD2 overexpression did not alter the somal distribution of control RGP cells but caused the vast majority of NDE1 knockdown RGP soma to accumulate at the ventricular surface. (d) Despite the accumulation of RGP soma at the ventricle in NDE1 knockdown with BicD2 overexpression, the mitotic index remained reduced. (e) Representative image of RGP cells with BicD2 overexpression along with double NDE1/NDEL1 knockdown, stained for PH3. Dashed line indicates ventricle. (f,g) Overexpression of BicD2 with NDE1/NDEL1 double knockdown fails to rescue the somal distribution pattern or mitotic index of double NDE1/NDEL1 knockdown RGP cells. (h,i) The same ratio of RGP nuclei were positive for CyclinD1 whether or not BicD2 was overexpressed along with the double NDE1/NDEL1 knockdown, indicating the prominence of the G1-to-S block in the double knockdown, and the G2 specificity of the BicD2 rescue strategy. Arrowheads mark electroporated RGP nuclei positive for CyclinD1. Dashed line indicates ventricle surface. Data are presented as scatterplot in c and f with bars representing the median±the interquartile range, and as mean±s.e.m. in d,g and i. Kolmogorov–Smirnov test for non-parametric distributions used in c and f (*P<0.05, n=407–591 RGP cells in c and n=421–487 RGP cells in f). Unpaired t-test used in d,g and i (*P<0.05, n=3 embryonic brains from different mothers). Scale bars, 10 μm. Also see Supplementary Fig. 7.

Figure 9. Roles of NDE1 and NDEL1 in the neural progenitor cell cycle.

Our data indicate that NDE1 is required for three distinct non-mitotic processes in the radial glia progenitor (RGP) cell cycle: G1-to-S progression (1), apical INM during late G2 (2) and G2-to-M transition (3). NDEL1 overexpression rescues (1) and (2), but not (3). Interference with the RGP cell cycle at any or all of these stages is proposed as an important contributor to microcephaly. Scale of image does not reflect actual duration of cell cycle stages.