McKeown CR and Cline HT (2019), Nutrient restriction causes reversible G2 arres...

XB-ART-56408

Development 2019 Oct 24;14620:. doi: 10.1242/dev.178871.

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Nutrient restriction causes reversible G2 arrest in Xenopus neural progenitors.

McKeown CR , Cline HT .

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Nutrient status affects brain development; however, the effects of nutrient availability on neural progenitor cell proliferation in vivo are poorly understood. Without food, Xenopus laevis tadpoles enter a period of stasis during which neural progenitor proliferation is drastically reduced, but resumes when food becomes available. Here, we investigate how neural progenitors halt cell division in response to nutrient restriction and subsequently re-enter the cell cycle upon feeding. We demonstrate that nutrient restriction causes neural progenitors to arrest in G2 of the cell cycle with increased DNA content, and that nutrient availability triggers progenitors to re-enter the cell cycle at M phase. Initiation of the nutrient restriction-induced G2 arrest is rapamycin insensitive, but cell cycle re-entry requires mTOR. Finally, we show that activation of insulin receptor signaling is sufficient to increase neural progenitor cell proliferation in the absence of food. A G2 arrest mechanism provides an adaptive strategy to control brain development in response to nutrient availability by triggering a synchronous burst of cell proliferation when nutrients become available. This may be a general cellular mechanism that allows developmental flexibility during times of limited resources.

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Species referenced: Xenopus laevis
Genes referenced: ins mtor sox2 tecta.2

Phenotypes: Xla.B + CldU labeling + feeding [+]

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	Nutrient restriction limits brain growth and NPC proliferation. (A) Diagram of the Xenopus optic tectum. NPCs reside along the ventricle at the midline (green), and their progeny are pushed laterally as NPCs divide and differentiate into neurons (red). The midline of the tectal ventricle is indicated with a dashed yellow line. (B) Experimental timeline for the data shown in C. Animals were treated for 24 h with CldU and then allowed to develop for 6 days in either fed or nutrient-restricted conditions. (C) Whole-mount confocal images of CldU immunofluorescence in the optic tectum in fed (i, iii) and nutrient-restricted (ii, iv) animals. 20× magnification 6.2 μm z-projections (i-ii) and 60× magnification 1.66 μm z-projections (iii-iv) are shown. Confocal images of brains were collected under identical settings and are shown at the same scale (i-ii and iii-iv). (D) Experimental timeline for the data shown in E. Animals were treated for 2 h with CldU and then allowed to develop for 2-3 days in either fed or nutrient-restricted conditions. (E) Whole-mount confocal images of Sox2 and CldU (i-ii), HuC/D and CldU (iii-iv), and HuC/D and pH3 (v-vi) immunofluorescence along the tectal midline in fed (i, iii, v) and nutrient-restricted (ii, iv, vi) animals. Single optical sections are shown. Scale bars: 100 μm (C); 20 μm (E).
	Nutrient-induced increase in cell proliferation occurs 16 h after feeding. (A) Experimental timeline. Animals were nutrient restricted for 2 days at Stage 47 and then food was re-introduced. Animals were sacrificed and processed for pH3 immunolabeling at the indicated time points after feeding. (B) Whole-mount confocal images of pH3 immunofluorescence in the optic tectum at the time points indicated. Fd, food. (C) Quantification of pH3 labeling showing total cell counts per tectum at each time point (mean±s.e.m.). Green data points indicate fed and gray are NR animals. At 16 h after feeding, pH3+ cells are significantly increased compared with NR controls, ****Pn=6-47 animals per group per time point, from multiple independent clutches, for a total of 304 animals, across 16 groups from multiple independent clutches; see Table S1. Scale bars: 100 μm.
	Nutrient-induced proliferation requires mTOR signaling. (A) Experimental timeline. Animals were nutrient restricted for 2 days after Stage 47 and then food was re-introduced concomitant with the mTOR-blocking drug rapamycin or DMSO vehicle for up to an additional 2 days. Brains were processed for pH3 immunolabeling 16 h, 24 h and 48 h after feeding, or for western blots 48 h after feeding as indicated. (B) Western blot analysis shows that feeding increased phospho-mTOR (p-mTOR) and that increase in p-mTOR was blocked by rapamycin. (C) Western blot analysis shows that feeding increased phospho-ribosomal protein S6 (p-rS6) and that increase was blocked by rapamycin. (D) Whole-mount confocal images of pH3 immunolabeling in the optic tectum 16 h, 24 h and 48 h after feeding, with or without rapamycin treatment. Fd, food. (E) Quantification of total pH3+ cells per tectum 16 h, 24 h and 48 h after feeding, with or without rapamycin showing that rapamycin blocks the feeding-induced increase in proliferation at all time points tests. Data for each condition are shown as individual data points and mean±s.e.m. are shown as black bars. Blue symbols signify rapamycin treatment and gray symbols are controls. For western blots, n=5 brains per treatment per time point, a minimum of 3 biological replicates was used for each quantification shown. For cell counts, n=8-18 animals per group per time point for a total of 150 animals across 12 groups from multiple independent clutches; see Table S1. **PPP
	Feeding triggers NPCs to exit G2 arrest and enter the cell cycle at M phase. (A) Experimental timeline. Stage 47 animals were nutrient restricted for 2 days and then treated with CldU for 24 h with or without food. After 24 h, brains were processed for both pH3 and CldU immunolabeling. (B,F) Whole-mount confocal images of CldU (red) and pH3 (green) immunolabeling in the optic tectum in nutrient-restricted (B) and fed (F) animals. Images are 60 μm z-projections. (C-E,G-I) Digitally magnified images from the midline of the animals shown in B and F. All samples were collected under identical imaging parameters and post-processed identically. (J) Quantification of pH3+ cells (green) and pH3+CldU+ (yellow) cells in the 60 μm Z-series showing that food increases the number of pH3+ cells but not the pH3+CldU+ population. (K) Fraction of pH3+ cells that had also been previously labeled with CldU presented as average of total cells per tectum ±s.e.m. n=7 animals per group; see Table S1. **PP
	Nutrient restriction increases DNA content in NPCs. (A-F) Whole tectum fluorescence intensity analysis. Brains from continuously fed (orange, A) and nutrient-restricted (blue, B) animals were stained with TOPRO-3 to label DNA. Boxes in A and B indicate the area analyzed by line scan. (C) Example of averaged line scans from four individual nutrient-restricted (blue) and three individual fed (orange) animals showing a higher peak of DNA labeling along the midline in the NR brains. (D) Average peak intensity measurements demonstrate increased fluorescence in the NR brains, consistent with increased DNA content. (E) Average area of TOPRO-3 labeling, indicating that there are more cells in the fed animals. (F) Fluorescent Index (ratio of peak intensity/area) indicates that DNA content is significantly greater in the tectal proliferative layer of nutrient-restricted animals than in fed animals. (G-I) Single cell fluorescent intensity analysis. Cell tracing of progenitor cells along the midline in fed (G) and nutrient-restricted (H) animals. Individually traced cells are outlined in yellow. Asterisk indicates an example neuronal cell body adjacent to the neuropil that was traced for baseline 2N DNA content. (I) Fluorescence/cell area ratio indicates that DNA content is higher in the progenitor population than in neurons (gray), and DNA content is further increased in NR-NPCs (blue) compared with fed NPCs (orange). For A-F, n=16-17 animals per group for a total of 33 animals from 2 independent clutches. For G-I, n=10 animals per group (50 cells per animal) for a total of 500 cells measured per condition. See Table S1 and Materials and Methods. ****P.
	Activation of insulin signaling is sufficient to push G2-arrested cells back into the cell cycle in the absence of nutrients. (A) Experimental timeline. Animals were nutrient restricted for 2 days and then 300 nM bPV(phen), or H2O control, was injected directly into the tectal ventricle. Brains were fixed and processed for pH3 immunofluorescence at 90 min and 4 h after injection. Control animals were nutrient restricted for 2 days and then fed for 16 h prior to fixation, as shown in the lower timeline. (B-D) Confocal images of pH3-labeled tecta from injected animals at 4 h and a control group that was given food to induce proliferation. (E) Quantification of pH3 labeling showing total cell counts per tectum in each condition (error bars represent s.e.m.). Gray data points are from NR and H2O-injected animals. Purple data points indicate bPV(phen) animals, and green data points represent fed controls. n=6-39 animals per group per time point for a total of 160 animals across 16 groups from multiple independent clutches, see Table S1. Fd, food. ****P

References [+] :

Acosta-Jaquez, Site-specific mTOR phosphorylation promotes mTORC1-mediated signaling and cell growth. 2009, Pubmed