Gibeaux R et al. (2018), Xenopus Hybrids Provide Insight Into Cell and O...

XB-ART-55549

Front Physiol 2018 Jan 01;9:1758. doi: 10.3389/fphys.2018.01758.

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Xenopus Hybrids Provide Insight Into Cell and Organism Size Control.

Gibeaux R , Miller K , Acker R , Kwon T , Heald R .

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Determining how size is controlled is a fundamental question in biology that is poorly understood at the organismal, cellular, and subcellular levels. The Xenopus species, X. laevis and X. tropicalis differ in size at all three of these levels. Despite these differences, fertilization of X. laevis eggs with X. tropicalis sperm gives rise to viable hybrid animals that are intermediate in size. We observed that although hybrid and X. laevis embryogenesis initiates from the same sized zygote and proceeds synchronously through development, hybrid animals were smaller by the tailbud stage, and a change in the ratio of nuclear size to cell size was observed shortly after zygotic genome activation (ZGA), suggesting that differential gene expression contributes to size differences. Transcriptome analysis at the onset of ZGA identified twelve transcription factors paternally expressed in hybrids. A screen of these X. tropicalis factors by expression in X. laevis embryos revealed that Hes7 and Ventx2 significantly reduced X. laevis body length size by the tailbud stage, although nuclear to cell size scaling relationships were not affected as in the hybrid. Together, these results suggest that transcriptional regulation contributes to biological size control in Xenopus.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: h1-10 hes7.2 ventx2.2

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	Figure 1. Growth and development of Xenopus le × ts viable hybrids. (A) Schematic of developmental outcomes of Xenopus laevis and Xenopus tropicalis cross-fertilization. (B) Agarose gel electrophoresis showing PCR amplification of 2 genomic loci in X. laevis (X. l, on chromosomes 5L and S) and one locus in X. tropicalis (X. t, chromosome 5). H1-10 indicates 10 randomly chosen hybrid tadpoles tested, confirming the consistent presence of all 3 subgenomes in hybrids. (C) Developmental timing in X. laevis and le × ts hybrid embryos. Average is plotted for each time point. Error bars show standard deviation. (D) Body length of tailbud stage X. laevis and le × ts hybrids. Box plots show all individual body lengths. Thick line inside box = average length, upper and lower box boundaries = ±standard deviation (SD). P-value was determined by two-tailed heteroscedastic t-test. Representative images of tailbuds at identical scale are shown on the right. (E) Body length of tadpoles throughout metamorphosis for X. laevis and le × ts hybrids. Average is plotted for each time point. Error bars show standard deviation. (F) Body length of X. laevis and le × ts hybrid froglets. Average is plotted for each time point. Error bars show standard deviation. Representative images of froglets at identical scale are shown on the right. (G) Size of erythrocyte cells and nuclei in X. laevis and le × ts hybrid adult frogs. Box plots show all individual cell or nuclear areas. Thick line inside box = average area, upper and lower box boundaries = ± SD. P-values were determined by two-tailed heteroscedastic t-test. Representative images of erythrocytes at identical scale are shown on the right.
	Figure 2. Nuclear to cell size relationships pre- and post-zygotic genome activation in le × ts hybrids compared to X. laevis diploids and haploids. (A) Schematic of generation of haploid X. laevis tadpoles via UV irradiation of sperm. (B) Developmental timing in X. laevis and haploid X. laevis embryos. Average is plotted for each time point. Error bars show standard deviation. (C) Body length of tailbud stage X. laevis and haploid X. laevis. Box plots show all individual body lengths. Thick line inside box = average length, upper and lower box boundaries = ± SD. P-value was determined by two-tailed heteroscedastic t-test. (D) Nuclear diameter vs. cell diameter in X. laevis, X. laevis haploid, and le × ts hybrid embryos. (E) Nuclear diameter vs. cell diameter in X. laevis, X. laevis haploid, and le × ts hybrid embryos at developmental stages 6 and 8. (F) Nuclear diameter vs. cell diameter in X. laevis, X. laevis haploid, and le × ts hybrid embryos at developmental stage 10. (G) Nuclear diameter vs. cell diameter in X. laevis, X. laevis haploid, and le × ts hybrid embryos at developmental stage 21. For (E–G), we ran an analysis of covariance (ANOCOVA test) to determine whether the nuclear to cell size scaling significantly depends on the embryo types. At stage 6, p = 0.132, at stage 8, p = 0.126, at stage 10, p = 2.558 × 10−6, and at stage 21, p = 1.110 × 10−7.
	Figure 3. Transcriptome analysis of le × ts hybrid embryos at the onset of zygotic genome activation. (A) Differential expression analysis of X. laevis maternal genes in stage 9 le × ts hybrid vs. X. laevis embryos. (B) Differential expression analysis of X. tropicalis paternal genes in stage 9 le × ts hybrid vs. X. laevis embryos. For both figures, RNA-seq reads are mapped to a database of combined X. laevis and X. tropicalis transcriptomes and significantly differentially expressed genes (DE; fold-change >2 and false discovery rate < 0.05) are marked in orange (see section Materials and Methods for more information).
	Figure 4. Organismal size in X. laevis embryos upon overexpression of candidate X. tropicalis transcription factors. (A) Workflow of candidate scaling factor screen. (B) Body length of tailbud stage injected X. laevis embryos. Thick line inside box = average length, upper and lower box boundaries = ±SD. Stars indicate the significance of the pooled results of 3 experiments with p < 0.05 (two-tailed heteroscedastic t-test). Red coloring indicates significance of each 3 individual technical replicates with p < 0.05 (two-tailed heteroscedastic t-test). Reduced number of measured embryos in Not is due to the fact that, overall, 67.5% of injected-embryos exogastrulated, indicating a developmental defect. (C) Representative images of injected X. laevis embryos 48 h post-fertilization. Hes7- (left) and Ventx2-injected (right) are shown (top) with corresponding controls (bottom). Images are at identical scale.

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