Acute Lengthening of Progenitor Mitosis Influences Progeny Fate during Cortical Development in vivo

Abstract

Background/aims: Prenatal microcephaly is posited to arise from aberrant mitosis of neural progenitors, which disrupts both neuronal production and survival. Although microcephaly has both a genetic and environmental etiology, the mechanisms by which dysregulation of mitosis causes microcephaly are poorly understood. We previously discovered that prolonged mitosis of mouse neural progenitors, either ex vivo or in vitro, directly alters progeny cell fate, -resulting in precocious differentiation and apoptosis. This raises questions as to whether prolonged progenitor mitosis affects cell fate and neurogenesis in vivo, and what are the underlying mechanisms?

Methods/results: Towards addressing these knowledge gaps, we developed an in vivo model of mitotic delay. This uses pharmacological inhibition to acutely and reversibly prolong mitosis during cortical development, and fluorescent dyes to label direct progeny. Using this model, we discovered that a causal relationship between mitotic delay of neural progenitors and altered progeny cell fate is evident in vivo. Using transcriptome analyses to investigate the state of delayed cells and their progeny, we uncovered potential molecular mechanisms by which prolonged mitosis induces altered cell fates, including DNA damage and p53 signaling. We then extended our studies to human neural progenitors, demonstrating that lengthened mitosis duration also directly alters neuronal cell fate.

Conclusions: This study establishes a valuable new experimental paradigm towards understanding mechanisms whereby lengthened mitosis duration may explain some cases of microcephaly.

Keywords: Cell fate; Cortex; Mitosis; Neurogenesis; Progenitor.

Figure 1.. A model for acute inducible prolonged mitosis in vivo.

(A) Experimental paradigm with injection of either DMSO or STLC at timepoint 0 and harvesting of brains for fate analysis between 8–73 hours later. (B) Immunofluorescence depicting PH3+ cells (red) and DAPI (white) at the ventricle of an E14.5 cortex, at different time points following injection of DMSO or STLC. Scale bar: 25 μm. White arrowheads: prometaphase cells; yellow arrowheads:- anaphase cells. (C) Quantification of the average number of prometaphase cells per 100 μm, along the ventricular surface of the cortex. N ≥ 3 embryos for each condition, at each timepoint. N=37 total embryos. Statistics: Anova analysis, post-hoc student’s t-test. ***, p=0.0005. ****, p<0.0001. Error bar=s.d.

Figure 2.. Effects of prolonged mitosis in vivo on cell fate acquisition.

(A) Scheme of injection paradigm used in this study. B) Time course of injection paradigm used to assess cell fate following prolonged mitosis. C, E, G) Images depicting E14.5 brains 16 hrs after injection with either STLC or DMSO and stained for C) Pax6 (white, red) (C) , Tbr2 (white, red) (E) , Neurod2 (white, red) (G) along with FlashTag (green). D, F, H) Quantification of the fraction of FT+ cells which are Pax6 (D), Tbr2 (F), and Neurod2 (G) 16h after injection of DMSO or STLC. Pax6 analysis DMSO: n=11 embryos; STLC: n=9 embryos, n=2 sections each. Tbr2 analysis, DMSO: n=10 embryos, STLC: n=9 embryos, n= 2 or more sections each. NeuroD2 analysis, DMSO: n=11 embryos, STLC: n=10 embryos, n=2 or more sections each. Statistics: student’s t-test. I) Images depicting FT (green), EdU (red), and NeuroD2 (purple) 26h post-injection of DMSO or STLC. J) EdU labeling scheme following injection of DMSO or STLC, analysis at 26h post-injection. K) Quantification of the fraction of FT cells which are EdU-NeuroD2+FT+ 26h after injection of DMSO or STLC. NeuroD2 **: p=0.0037. Pax6 **** p<0.0001. EdU/NeuroD2 *: p=0.04. Scale bars: A: 20 μm. E,C, G, 25 μm; J, 100 μm. Error bar=s.d.

Figure 3.. Prolonged mitosis in vivo results in apoptosis in daughter cells.

A) Images depicting the VZ/SVZ with FT (green) and CC3 (white) 8h after injection of DMSO or STLC. B) Quantification of the fraction of FT cells which are CC3+FT+ cells in 200 μm wide radial columns. C) Quantification of CC3+ cells that are FT+ at 8h in STLC condition. Dots represent individual embryos, n= 2 or more sections each. D) Images depicting the VZ/SVZ +16h after injection of STLC, depicting FT (green), CC3 (purple), and Tuj1 (red). Inset, right: ROI depicting CC3 and Tuj1. White arrowheads: FT+CC3+Tuj1+; Yellow arrowheads: FT+CC3+Tuj1−; Blue arrowheads: FT+CC3+Tuj1−. E) Quantification of the fraction of CC3+ cells that are either Tuj1+ or Tuj1− 16h after STLC injection, in 200 μm wide radial columns. N>10 sections. F and G) Quantification of the fraction of FT+ cells which are Pax6 (F) or NeuroD2 (G) 16h after injection of DMSO or STLC in E14.5 Bax−/− embryos; in 200 μm wide radial columns. Pax6 analysis, DMSO: n=4 embryos; STLC: n=5 embryos, n=3 sections each. NeuroD2 analysis, DMSO: n=5 embryos, STLC: n=6 embryos, n= 3 sections each. H) Comparison of fold changes in expression of Pax6 or NeuroD2 between WT and Bax−/− embryos at 16h following STLC injection. CC3 8h ***: p=0.001, 8h v 16h: p=0.05. BAX Pax6 *: p=0.015, NeuroD2 *: p=0.03. Pax6 BAX v WT *: p=0.01. Students t-test. Scale bars: A, D, 25 μm; D, 10 μm, Error bar=s.d.

Figure 4.. Transcriptome analysis following prolonged mitosis.

A) Schematic of RNA-seq analysis. B) Images depicting cortices stained for FT (white) 2 and 9 hrs after injection with either DMSO and STLC conditions, corresponding to RNA-seq timepoints. C, D) Cell cycle plots of FACS sorted populations by condition and timepoint. Y axis: cell count, X axis: Propidium Iodide. E) Graph of total significant transcript changes at both timepoints, >2-fold expression difference STLC v DMSO, p<0.05. F, G) Scatterplots of gene expression changes at +2h (F) and +9h (G) timepoints. Red: p<0.05, enriched in STLC. Blue: p<0.05, enriched in DMSO. Transcripts of notable change or associated with significant pathways have been annotated. H) qPCR for select RNA-seq candidates at +2h and +9h timepoints. Neurog1 ***: p=0.004, Tcf19 ***: p=0.02, Ccng1 ***: p<0.0001, Cdkn1a ***: p=0.0005. Dots equal independent biological replicates. At +2h, samples were identical to those use for FACs, whereas at +9h, 3 brains were from FACS and remaining were independent replicates. Error bar=s.d.

Figure 5.. Upregulated P53 pathway and DNA damage following prolonged mitosis.

A) Images of the VZ/SVZ depicting FT (green) and p53 (white) 8h after injection of DMSO or STLC. b) Quantification of the fraction of FT+ cells that are CC3+FT+ cells 4h, 6h, and 8 h after STLC injection via analysis of 200micron cortical columns. Dots represent individual embryos, average of 2 or more sections per embryo. 6h**: p=0.004, 8h ****: p<0.0001, 10h ***: p=0.0003, 6h v 8h *:p=0.02, 8h v 10h: p=0.04. C) Quantification of the fraction of FT+ cells that are p53+ at 8h in STLC condition. C) Percent of p53+ cells labeled by FlashTag 8h after STLC injection. N=3 embryos, average of 3 sections per embryo. E) Images of H2AX (red) and DAPI (white) in mitotic cells at the ventricle 3h after injection of DMSO or STLC. F) Quantification of H2AX intensity of mitotic cells at the ventricle 3h after DMSO or STLC injection. Dots represent individual cells across two or more embryos. Note for STLC, primarily FT+ cells were quantified in order to enrich for mitotic cells. Statistics: student’s t-test. metaphase ****: p<0.0001. anaphase/telophase ****: p<0.0001. Scale bar: A, 25 μm, E, 5 μm, Error bar= s.d.

Figure 6.. Prolonged mitosis in human neural progenitors directly alters cell fate.

A, B) Panels of live imaging timeseries depicting neurogenic (A) and proliferative divisions (B) with immunostaining for Tuj1 (red) and DIC. Arrowheads indicate 1 cell over time which divides to become 2 cells. C) Quantification of average mitosis duration for NPCs treated with either DMSO (blue) or STLC (red). Student’s t-test. ****: p<0.0001. D) Histogram of NPC mitosis duration, using 50min bins. Chi-square, ****: p<0.0001. E) Quantification of fraction of NPC divisions which are proliferative (Tuj1−; green), asymmetric neurogenic (1 Tuj1−, 1Tuj1+, white), or symmetric neurogenic (both Tuj1+, black), under different conditions and graphed by mitosis duration. DMSO: n=74 daughter pairs, STLC <50 mins: n=58 daughter pairs, STLC >50 mins: n= 41 cells. Chi-square with Bonferroni correction followed by post-hoc analysis. DMSO v STLC delayed **: p=0.006, STLC normal v delayed **: p=0.009, post hoc proliferative fates **: p=0.001, asymmetric neurogenic **: p=0.003. F) Histogram of neurogenic divisions (% of total at each mitotic duration) in 50 min bins. Chi-square ****: p<0.0001. G) Quantification of the fraction of NPC divisions generating viable (black) or apoptotic (white) progeny. DMSO: n=74 daughter pairs, STLC <50 mins: n=58 daughter pairs, STLC >50 mins: n= 66 cells. Statistics: Chi-square and post-hoc chi-square with Bonferroni correction. DMSO v Delayed STLC ****: p<0.0001, STLC normal v delayed STLC ****: p<0.0001. H) Histogram of proportion of apoptosis in progeny. ANOVA *: p=0.02, Error bar=s.d., Scale Bar= A, B: 20 μm.