January 1, 2018;
Paternal chromosome loss and metabolic crisis contribute to hybrid inviability in Xenopus.
Hybridization of eggs and sperm from closely related species can give rise to genetic diversity, or can lead to embryo inviability owing to incompatibility. Although central to evolution, the cellular and molecular mechanisms underlying post-zygotic barriers that drive reproductive isolation and speciation remain largely unknown. Species of the African clawed frog Xenopus provide an ideal system to study hybridization and genome evolution. Xenopus laevis is an allotetraploid with 36 chromosomes that arose through interspecific hybridization of diploid progenitors, whereas Xenopus tropicalis is a diploid with 20 chromosomes that diverged from a common ancestor approximately 48 million years ago. Differences in genome size between the two species are accompanied by organism size differences, and size scaling of the egg and subcellular structures such as nuclei and spindles formed in egg extracts. Nevertheless, early development transcriptional programs, gene expression patterns, and protein sequences are generally conserved. Whereas the hybrid produced when X. laevis eggs are fertilized by X. tropicalis sperm is viable, the reverse hybrid dies before gastrulation. Here we apply cell biological tools and high-throughput methods to study the mechanisms underlying hybrid inviability. We reveal that two specific X. laevis chromosomes are incompatible with the X. tropicalis cytoplasm and are mis-segregated during mitosis, leading to unbalanced gene expression at the maternal to zygotic transition, followed by cell-autonomous catastrophic embryo death. These results reveal a cellular mechanism underlying hybrid incompatibility that is driven by genome evolution and contributes to the process by which biological populations become distinct species.
R35 GM118183 NIGMS NIH HHS
, R01 CA172667 NCI NIH HHS
, 4T32GM007232-40 NIH HHS
, R01 HD069344 NICHD NIH HHS
, T32 GM007232 NIGMS NIH HHS
, S10 OD018174 NIH HHS
, DP1 GM106408 NIGMS NIH HHS
, R35 GM122480 NIGMS NIH HHS
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Figure 1. a, Schematic of X. laevis and X. tropicalis cross-fertilization outcomes. b, Developmental timing in X. tropicalis and te × ls hybrid embryos. Average is plotted for each time point. Error bars, s.d. c, Representative images of X. tropicalis and te × ls hybrid embryos at stages 3 and 10 from experiments in b (n = 16 X. tropicalis and n = 16 te × ls hybrid embryos from four independent experiments). Arrow indicates vegetal cells where death initiates. d, Schematic of animal cap assay and images of at 2, 9, and 16 h after isolation. Six animal caps were imaged and identical results were obtained in three different experiments. Scale bars in c and d, 200 μm. e, Images showing haploid phenotype following fertilization of X. tropicalis eggs with ultraviolet-irradiated sperm. Identical results were observed in n = 3 experiments. f, Time-lapse images of dividing cell in a te × ls hybrid animal cap (Supplementary Video 5). Arrow indicates a mis-segregated chromosome. Mis-segregated chromosomes were observed in n = 3 live te × ls hybrid animal caps in three experiments. Time is in minutes:seconds. g, Immunofluorescence images showing chromosome bridges, mis-segregated chromosomes, and micronuclei throughout te × ls hybrid embryos. Scale bars in f and g, 10 μm. Quantification of n = 81 X. tropicalis and n = 78 te × ls hybrid anaphases in n = 17 and 16 embryos, respectively, from four datasets obtained from three experiments presented as averages ± 1 s.d., show a significant difference by Fisher’s 2 × 3 contingency test (P = 0). Quantification of micronuclei in te × ls hybrid embryos is detailed in Extended Data Fig. 1b.
Figure 2. a, Fluorescence images of spindles formed around X. tropicalis, le × ts hybrid, and X. laevis chromosomes in X. tropicalis egg extract. Scale bar, 10 μm. Quantification for n = 147, 103, and 156 spindles quantified for X. tropicalis, le × ts hybrids, and X. laevis embryo nuclei, respectively, from three different egg extracts, is presented in Extended Data Fig. 1e. b, Fluorescence images of X. laevis chromosomes stained for CENP-A or Ndc80 following replication in X. laevis or X. tropicalis egg extract. CENP-A and Ndc80 labelling was quantified from six experiments (three biological replicates in two technical replicates), a total of n = 1,792 and n = 1,959 chromosomes, respectively, in X. laevis extract, and n = 2,692 and n = 1,930, respectively, in X. tropicalis extract. Scale bars, 5 μm. Box plots show the six experiment percentages as individual data points, their average as thick lines, and 1 s.d. as grey boxes. Ninety-five per cent confidence intervals are 96.2 ± 1.9% in X. laevis extract compared with 82.7 ± 5.7% in X. tropicalis extract for CENP-A, and 83.5 ± 6.1% compared with 71.1 ± 6.0% for Ndc80. P values were determined by two-tailed heteroscedastic t-test. c, Circle plot of whole-genome sequencing data for te × ls hybrid embryos aligned and normalized to the genomes of X. tropicalis (blue) and X. laevis (green), with underrepresented genome regions in black. d, Expanded view of chromosome (Chr.) 3L and 4L breakpoints with deleted regions (Del.) indicated in two biological replicates (Rep.).
Figure 3. a, Schematic of polar body suppression experiment and images of tte × ls rescued embryos 24 and 48 h.p.f. A total of nine tte × ls embryos were obtained in four different experiments. b, Box plot of nuclear sizes (n = 988 nuclei from three tte × ls embryos and n = 777 from three X. tropicalis embryos at stage 21) showing the average area as thick lines and 1 s.d. as grey boxes. Ninety-five per cent confidence intervals are 98.1 ± 2.2 μm2 for tte × ls and 78.0 ± 1.7 μm2 for X. tropicalis embryos. P values were determined by two-tailed heteroscedastic t-test. c, Levels of 179 metabolites in X. tropicalis and te × ls hybrid embryos 7 h.p.f. Levels were obtained from five samples from three independent fertilizations, each averaged and plotted as log2 of the ratio with the control (see Methods). P values were calculated using a two-tailed homoscedastic t-test. The average and 1 s.d. for the differentially represented metabolites are shown, and 95% confidence intervals given in Extended Data Fig. 3b. d, Differential gene expression between te × ls and te × ts (see Methods). All detected transcripts (n = 8,379) are plotted in blue. Transcripts corresponding to genes lost from chromosomes 3L and 4L (n = 270) are plotted in green. e, Differential expression of metabolism genes between te × ls and te × ts (see Methods). Differentially expressed metabolism transcripts (n = 165) are plotted in orange, all detected transcripts (n = 8,379) in blue (top), and differentially expressed metabolism transcripts located on chromosomes 3L and 4L (n = 35) in green (bottom).