Cadart C et al. (2023), Polyploidy in Xenopus lowers metabolic rate by ...

XB-ART-59762

Curr Biol 2023 May 08;339:1744-1752.e7. doi: 10.1016/j.cub.2023.03.071.

Show Gene links Show Anatomy links

Polyploidy in Xenopus lowers metabolic rate by decreasing total cell surface area.

Cadart C , Bartz J , Oaks G , Liu MZ , Heald R .

???displayArticle.abstract???
Although polyploidization is frequent in development, cancer, and evolution, impacts on animal metabolism are poorly understood. In Xenopus frogs, the number of genome copies (ploidy) varies across species and can be manipulated within a species. Here, we show that triploid tadpoles contain fewer, larger cells than diploids and consume oxygen at a lower rate. Drug treatments revealed that the major processes accounting for tadpole energy expenditure include cell proliferation, biosynthesis, and maintenance of plasma membrane potential. While inhibiting cell proliferation did not abolish the oxygen consumption difference between diploids and triploids, treatments that altered cellular biosynthesis or electrical potential did. Combining these results with a simple mathematical framework, we propose that the decrease in total cell surface area lowered production and activity of plasma membrane components including the Na+/K+ ATPase, reducing energy consumption in triploids. Comparison of Xenopus species that evolved through polyploidization revealed that metabolic differences emerged during development when cell size scaled with genome size. Thus, ploidy affects metabolism by altering the cell surface area to volume ratio in a multicellular organism.

???displayArticle.pubmedLink??? 37080197
???displayArticle.pmcLink??? PMC10184464
???displayArticle.link??? Curr Biol
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: cavin2
GO keywords: chromosome [+]
???displayArticle.antibodies??? Tjp1 Ab2

???attribute.lit??? ???displayArticles.show???

	Graphical Abstract.
	Figure 1. Triploid X. laevis develop normally (A) Triploid X. laevis were obtained by blocking polar body extrusion post fertilization (pf) using a cold shock. (B) The 1.5-fold increase in chromosome number in triploid (3N) compared with diploid (2N) embryos was verified by metaphase chromosome spreads. Scale bars, 10 μm. (C) Representative images of diploid and triploid embryos obtained from the same clutch of eggs. Scale bars, 1 mm. (D) Compared with diploids, triploids showed a comparable mass increase except for a transient ∼5% reduction during day 3 (19 clutches, n = 10–82 embryos per ploidy and stage; details in Table S1). (E) TMR-phalloidin staining of actin in ventral epithelial cells of stage 41 diploid and triploid X. laevis embryos showing cell outlines and strong actin enrichment at the surface of multiciliated cells. (F) Area of diploid and triploid multiciliated cells in embryos at stages 33–34, 37–38, and 41, normalized to diploids of each clutch. (3 clutches, n > 150 for each condition). (D and F) Welch two-sample t test comparing the means, ∗p < 0.5, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S1.
	Figure 2. Metabolism assessed by single-tadpole oxygen consumption rates varies between diploids and triploids of both X. laevis and X. borealis (A) Single tadpoles were placed in individual glass vials sealed from air. The decrease in O2 levels over time was used to calculate the oxygen consumption rate (OCR) for each tadpole. (B) OCR as a function of body mass in diploid X. laevis embryos from day 2 to 5 pf. The curve shows a fit with values and (n = 271) (see STAR Methods and Table S2). (C) OCR for bins of body mass and stages in X. laevis (left) and X. borealis (right) of diploid and triploid embryos (t test comparing the means, ∗p < 0.5, ∗∗p < 0.01; 3–5 clutches per condition; details in Table S3). (D) OCR as a function of body mass in diploid (n = 189) and triploid (n = 165) tadpoles from day 3 (stage 41) to day 5 (stage 46). The lines represent linear fits of the logarithmic values, which yielded the same allometric scaling component exponent α ( ), but different Y-intercepts that indicate a lower basal metabolic rate (B0) in triploids (see STAR Methods and Table S2). See also Figure S2.
	Figure 3. An embryo energy budget establishes the costs of proliferation, biosynthesis, and maintenance (A) OCR per mass as a function of palbociclib doses for stage 41 diploid embryos and two bins of mass (6 clutches, n = 2–57 per bin of mass and palbociclib concentration). (B) Representative image of phospho-histone H3 immunostaining and Hoescht DNA dye staining of tadpole tails (scale bar, 200 μm). (C) Mitotic cell density in the tails of stage 41 diploid embryos assessed by quantifying phospho-histone H3-positive cells (n = 2, 5–6 embryos per clutch, Welch two-sample t test comparing the means, ∗∗∗p < 0.001). (D) OCR per mass for stage 41 diploid embryos in water, 6 μg/mL palbociclib, 12.5 nmol/mL torkinib, 1 μg/mL torin-1, 0.1% DMSO, or 200 μg/mL ouabain. (E) The percentage decrease of OCR in diploids upon treatment with drugs in (D) provides an estimate of the contribution of proliferation (palbociclib), growth (torkinib, torin-1), and maintenance (ouabain) to the overall embryo energy expenditure. See also Figures S3 and S4.
	Figure 4. The energetic cost of cell membrane maintenance, not proliferation, accounts for the difference in metabolism between diploids and triploids (A) Mitotic cell density in the tails of diploid and triploid embryos assessed by quantifying phospho-histone H3-positive cells (n = 3, 5–6 embryos per clutch). (B) OCR per mass normalized to the median diploid value for each condition in stage 41 tadpoles during a 6-h incubation in water, 6 μg/mL palbociclib, 12.5 nmol/mL torkinib, 0.1% DMSO, or 200 μg/mL ouabain. Results are shown for 3–4 mg embryos except 5–6 mg for ouabain (n = 3–5 clutches per condition; details in Table S4). Dashed line indicates the average in untreated triploids. Welch two-sample t test comparing the means, ∗p < 0.5, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S4.
	Figure 5. Metabolic rates of Xenopus species scale with cell size, not ploidy (A) X. longipes, X. laevis, and X. tropicalis are 12N, 4N, and 2N, respectively, with corresponding differences in chromosome (ch.) number; tree not to scale with phylogenetic distances. (B) Cell size measurements show scaling with genome size at stage 48, but not stage 41 (2–5 clutches, n > 40 per condition, error bars represent the standard deviation, X. tropicalis and X. longipes stage 48 data from Miller et al.23). (C) OCR across species: (top) before the onset of cell size scaling with genome size at day 3–4 (stage 41–46) and (bottom) after scaling onset (stage 48–50) at day 6–8 (X. laevis), 7–13 (X. longipes), and 26–46 (X. tropicalis) (see Table S5 for n of each bin). (D) OCR data at stages 48 or later (from C, bottom) normalized to the mean X. laevis diploid value for each bin of mass (each dot is the value for a different bin of mass and species) and plotted as a function of mean cell area at stage 48 (B) normalized to the X. laevis diploid value. (B and D) ρ is the Pearson’s correlation coefficient of the means weighted by the SD (∗p < 0.05, ∗∗p < 0.01). See also Figures S2 and S5.
	Figure S1. Triploid embryos develop and survive at rates comparable to diploids. Related to Figure 1. (A)Survival rate over the first 7 days post fertilization for two representative clutches. Each line represents a technical replicate. (B) Developmental curves of diploids and triploids at 24oC (three clutches containing diploids and triploids). The line represents a polynomial fit. (C) Area of diploid and triploid goblet-like cells in embryos at stages 33-34, 37-38 and 41, normalized to diploids of each clutch. (3 clutches, n>68 for each condition). Welch two sample t-test comparing the means, : p<0.5, : p<0.01,**: p<0.001.
	Figure S2. Quality controls and details of OCR experiments. Related to Figures 2 and 5. (A)Example of measurements obtained using a blank vial containing no tadpoles. Blanks were used to assess the time at which O2 and temperature stabilized for each experiment. Timepoints acquired before equilibration were discarded. Details of the coefficient of variation (CV) of O2 (B) and temperature (C) in the blanks of each of the 66 experiments used for Figures 2B-D and 5CD show that these variables were very stable once the timepoints prior to equilibrium were removed. (D) Average temperature showed little variation across all experiments. Details of the duration (E), R2 (F), and p values (G) of all 1401 individual linear fits performed to calculate singleembryo or single-tadpole OCR for all 66 experiments used for Figures 2B-D and 5C-D. (H) Examples of two OCR experiments during which the embryo died showing that there is no subsequent O2 consumption, suggesting a negligible contribution of bacteria to the OCR measurements. (I) Comparison of OCR normalized to mass and binned by mass and stage over 5 days of development reveals a transient increase followed by consistent decrease in metabolic rate in X. laevis triploids compared to diploids (3 to 5 clutches per condition, details in Table S3). (J)OCR and subsequent sex genotype using PCR was performed on diploid X. laevis stage 41. X.laevis triploids are all females due to the way they are obtained. Comparison of OCR in male and female X. laevis diploids reveals no sex-related metabolic differences, indicating that this was not a cause of the OCR decrease in triploids. (K) Normalization to diploid animals indicates that both X. borealis and X. laevis have a ~6% decrease in metabolic rate.
	Figure S3. Controls for the drug treatments used to establish the embryo energy budget at stage 41. Related to Figure 3. (A)Tail length throughout development (2-4 clutches per stage, n > 30 embryos per ploidy and stage). (B) OCR per mass as a function of torin-1 (left) and torkinib (right) doses for stage 41 diploid embryos and two bins of mass. Torkinib doses higher than 12.5 nmol/mL induced significant death rate and were thus removed from the analysis (torin-1: 3 clutches, n = 4-22 per bin of mass and dose; torkinib: 2 clutches, n = 2-20 per bin of mass and dose). (C) OCR per mass as a function of ouabain doses for stage 41 diploid embryos and three bins of mass (6 clutches, n = 5-26 tadpoles per bin of mass and dose). (D) The 6-hr treatment with ouabain induced a significant increase in embryo body mass compared with embryos treated with palbociclib or DMSO. This is likely because of perturbations of intracellular osmolarity following inhibition of the Na+/K+ ATPase. (E) Details of results from Figure 3D showing OCR/mass in diploids for each pairof control (drug solvent) and drug treatment. Palbociclib and ouabain were solvated in water, torkinib in ethanol (final ethanol concentration at 0.025%), and torin-1 in DMSO (final DMSO concentration at 0.1%). The solvents did not affect the average OCR/mass value compared with embryos in water. Palbociclib experiments displayed a slightly higher control value (see Figure S4A).
	Figure S4. Normalization strategy for calculating the embryo energy budget and controls for drug treatments. Related to Figures 3 and 4. (A) The average OCR measured in diploids was higher in untreated controls (in water) for the set of experiments performed in the palbociclib assay (Figure 3A and D) compared with previous measurements in water (Figure 2C). This may be due to a transient problem in calibration of the SDR device since later experiments in water, ethanol, or DMSO (Figure 3D and S3E) showed that diploids were again at the initially measured value. (B) To account for this control OCR variability and to calculate the OCR decrease with drug treatments in Figure 3E, we first normalized each drug-treated condition to the mean diploid untreated value for that experiment. (C) Mitotic cell density in tails after 6 hr treatment with or without 6 µg/mL palbociclib (2 clutches, 5-6 embryos per clutch and condition; Welch t-test comparing the mean, : p<0.05, *: p<0.01, n.s.: not significant). (D) Experiments with torkinib (right) and solvent control (left) showing that 0.025% ethanol does not impact the OCR decrease in stage 41 triploids compared to diploids.
	Figure S5. Details of cell size and mass measurements across species. Related to Figure 5. (A)Area of goblet-like and multicilliated cells measured as in Figure 2A-B at stage 41 and at stage 48 (data for X. tropicalis and X. longipes at stage 48 come from ref.S1). (B) Distribution of tadpole mass across species shows that X. laevis and X. longipes have overlapping masses during week 2 while X. tropicalis did not. In Figure 5C-D, to compare tadpoles of similar mass, X. tropicalis from week 5-7 are compared with X. laevis and X. longipes from week 2 (see also Table S5). (C)Same as in Figure 5C but with the largest bin of mass, 11-12 mg. X. laevis diploids displayed a lower OCR at this mass for unexplained reasons, but otherwise trends among X. tropicalis, triploid X. laevis and X. longipes appeared qualitatively similar to that observed for all other mass bins.

References [+] :

Atkins, Effect of acclimation temperature on routine metabolic rate in triploid salmonids. 2008, Pubmed