Narbonne P et al. (2011), Deficient induction response in a Xenopus nucle...

XB-ART-44545

PLoS Biol 2011 Nov 01;911:e1001197. doi: 10.1371/journal.pbio.1001197.

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Deficient induction response in a Xenopus nucleocytoplasmic hybrid.

Narbonne P , Simpson DE , Gurdon JB .

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Incompatibilities between the nucleus and the cytoplasm of sufficiently distant species result in developmental arrest of hybrid and nucleocytoplasmic hybrid (cybrid) embryos. Several hypotheses have been proposed to explain their lethality, including problems in embryonic genome activation (EGA) and/or nucleo-mitochondrial interactions. However, conclusive identification of the causes underlying developmental defects of cybrid embryos is still lacking. We show here that while over 80% of both Xenopus laevis and Xenopus (Silurana) tropicalis same-species androgenetic haploids develop to the swimming tadpole stage, the androgenetic cybrids formed by the combination of X. laevis egg cytoplasm and X. tropicalis sperm nucleus invariably fail to gastrulate properly and never reach the swimming tadpole stage. In spite of this arrest, these cybrids show quantitatively normal EGA and energy levels at the stage where their initial gastrulation defects are manifested. The nucleocytoplasmic incompatibility between these two species instead results from a combination of factors, including a reduced emission of induction signal from the vegetal half, a decreased sensitivity of animal cells to induction signals, and differences in a key embryonic protein (Xbra) concentration between the two species, together leading to inefficient induction and defective convergence-extension during gastrulation. Indeed, increased exposure to induction signals and/or Xbra signalling partially rescues the induction response in animal explants and whole cybrid embryos. Altogether, our study demonstrates that the egg cytoplasm of one species may not support the development promoted by the nucleus of another species, even if this nucleus does not interfere with the cytoplasmic/maternal functions of the egg, while the egg cytoplasm is also capable of activating the genome of that nucleus. Instead, our results provide evidence that inefficient signalling and differences in the concentrations of key proteins between species lead to developmental defects in cybrids. Finally, they show that the incompatibilities of cybrids can be corrected by appropriate treatments.

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Species referenced: Xenopus laevis
Genes referenced: cripto.3 inhba prkaa1 ran tbxt vegt

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	Figure 1. Early development of Xenopus androgenetic haploids, hybrids, and cybrids. show more (Aï¿½D) Haploid development frequently gives rise to stunted swimming tadpoles in both X. laevis and X. tropicalis. Typical stage 38 (A) lxl, (B) [l]xl (lx[l] were identical (unpublished data)), (C) txt, and (D) [t]xt embryos are shown. (E) Haploid X. laevis development is improved by the addition of a X. tropicalis sperm nucleus. A typical stage 38 lxt hybrid is shown. (Fï¿½G) [l]xt cybrids have a reduced developmental capacity compared to [l]xl or [t]xt and never form swimming tadpoles. Two of the most developmentally advanced [l]xt cybrid postneurulae after ~72 h at 23ï¿½C are shown. Substantial posterior axis elongation such as in the individual shown in (G) is very rare and occurs in less than 1% of [l]xt cybrids. Scale bars in (Aï¿½B, Eï¿½G) and (Cï¿½D): 1 mm. doi:10.1371/journal.pbio.1001197.g001
	Figure 4. Reduced elongation of stage 10.5 dorso-marginal explants in cybrid embryos. show more Dorso-marginal explants were dissected from stage 10.5 (A) lxl, (B) [l]xl, (C) txt, (D) [t]xt, and (E) [l]xt embryos and cultured overnight in 1ï¿½ MBS. The axis of elongation (length) was highlighted in red and the width, in green. Five representative conjugates are shown for each experiment; see Table 2 for total numbers. Scale bars in (A, B, E) and (C, D): 0.5 mm. doi:10.1371/journal.pbio.1001197.g004
	Figure 5. Elongation defects of cybrids result from deficient emission of, and response to, induction signals. show more Stage 8ï¿½9 animal caps (black inset) were dissected from (Aï¿½D) lxl or from (Eï¿½H) [l]xt cybrid embryos and conjugated to same-stage vegetal halves (white inset) of the following kinds of embryos (A, E) lxl, (B, F) txt, (C, G) [t]xt, and (D, H) [l]xt. Animal-vegetal conjugations were cultured overnight in MBS. The axis of elongation, where present, was highlighted in red. 3ï¿½6 representative conjugates are shown for each experiment; see Table 3 for total numbers. Scale bars in (A, D, E, H) and (B, C, F, G): 1 mm. doi:10.1371/journal.pbio.1001197.g005
	Figure 6. Activin-induced animal cap elongation is defective in cybrid embryos. show more Animal caps dissected from stage 8 (A) lxl, (B) [l]xl, (C) txt, (D) [t]xt, and (E) [l]xt embryos were cultured either directly in MBS (left), or in MBS supplemented with 5 ng/ml Activin for 1 h, washed, and transferred to MBS overnight (right). (Fï¿½G) Induction and elongation response of [l]xt cybrid embryo animal caps are improved if (F) Activin exposure is doubled and improved further if (G) Xbra is also overexpressed. The axis of elongation (length) is highlighted in red and the width, in green. 4ï¿½7 representative explants are shown for each experiment; see Table 4 for total numbers. Scale bars in (A, B, E, Fï¿½G) and (C, D): 1 mm. doi:10.1371/journal.pbio.1001197.g006
	Figure 7. Increased induction signalling partially rescues convergence-extension in whole cybrid embryos.Injection of (A, C) human recombinant Activin A protein into the blastocoel of stage 8, or (B, D) FRL-1 mRNA in the animal half of one-cell [l]xt cybrid embryos, significantly improved (A, B) blastopore closure and (C, D) embryo elongation. Three representative H2O control (left) or Activin/FRL-1-treated (right) embryos are shown below each graph. The blastopore closure ratio (blastopore area/embryo area) was calculated from the respective diameters of the blastopore and embryo at 22 h post-fertilization. The embryo elongation ratio was obtained by dividing the embryo length by the greatest of the height or width at 32 or 46 h post-fertilization. Error bars represent the standard deviation between the means of 4 (Activin) or 3 (FRL-1) replicate experiments. p values were obtained using the one-tailed t test. n, sample size. Scale bar: 1 mm.
	Figure 2. EGA and translation appear normal in cybrid embryos.(A) Transcription of key embryonic genes is normal in [l]xt cybrids. Real-time RT-PCRs were carried out to compare relative mRNA/total RNA quantities for the indicated genes in stage 10.25 [l]xt cybrid embryos as compared to that in txt. Relative mRNA quantities were found to be identical for Vegt and Xbra at stage 10.25 between txt and [t]xt (unpublished data). Error bars represent the standard deviation between three to six replicate experiments, each of which is the average of three to six single embryos. No significant difference was found (p>0.05; two-tailed t test) for any of the genes tested. (B) Xbra protein is synthesized normally from the X. tropicalis genome by the X. laevis cytoplasm. Western blot analysis was carried out to evaluate Xbra protein levels in stage 11 embryos of the indicated combinations. Â§ [l]xt embryos never morphologically reach stage 11, but they were harvested when [l]xl siblings reached stage 11. Ran was used as a loading control as it is present at the same concentration in the eggs of both species [67]. (C) Quantification of pixel intensity indicates a significant (â¼2-fold) increase in Xbra protein concentration at stage 11 in txt relative to lxl (pâ=â0.014; one-tailed t test with unequal variance; nâ=â4).
	Figure 3. Cybrid early gastrulae are not energy-deficient.(A) ATP content, measured using a luciferase-based assay, is normal in cybrid embryos. Samples were collected every 3 h, except for the interval between stage 8.5 and 10.25 for [l]xl and [l]xt, which was about 4 h. Each point on the graph represents the average measurement (triplicate readings) of two pools of five embryos, all from the same X. laevis female. Error bars represent the standard deviation between the average readings of the two pools. (B) The activity of a key energy stress-sensing kinase is normal in [l]xt cybrids. Activated AMPK was detected on a Western blot using monoclonal anti-phospho-AMPK antibodies. Oligomycin, an inhibitor of mitochondrial ATP synthesis [46], was used as a positive control to induce energy stress. Ran was used as a loading control [67]. All X. laevis egg-based embryos are from the same female; X. tropicalis egg-based embryos are from two different females, which might explain the slight difference in AMPK phosphorylation observed between the two samples. * Non-specific band present in the eggs of both X. laevis and X. tropicalis. â Oligomycin-treated embryos (40 ÂµM) arrested development at stage 9, but were harvested when diploid controls reached stage 11. Â§ [l]xt embryos morphologically never reach stage 11, but they were harvested when their [l]xl control siblings reached stage 11.

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Beyhan, Interspecies nuclear transfer: implications for embryonic stem cell biology. 2007, Pubmed