Altering the levels of nuclear import factors in early Xenopus laevis embryos affects later development

Abstract

More than just a container for DNA, the nuclear envelope carries out a wide variety of critical and highly regulated cellular functions. One of these functions is nuclear import, and in this study we investigate how altering the levels of nuclear transport factors impacts developmental progression and organismal size. During early Xenopus laevis embryogenesis, the timing of a key developmental event, the midblastula transition (MBT), is sensitive to nuclear import factor levels. How might altering nuclear import factors and MBT timing in the early embryo affect downstream development of the organism? We microinjected X. laevis two-cell embryos with mRNA to increase levels of importin α or NTF2, resulting in differential amounts of nuclear import factors in the two halves of the embryo. Compared to controls, these embryos exhibited delayed gastrulation, curved neural plates, and bent tadpoles with different sized eyes. Furthermore, embryos microinjected with NTF2 developed into smaller froglets compared to control microinjected embryos. We propose that altering nuclear import factors and nuclear size affects MBT timing, cell size, and cell number, subsequently disrupting later development. Thus, altering nuclear import factors early in development can affect function and size at the organismal level.

Fig 1. Differential levels of nuclear import factors in the two halves of an early embryo affect gastrulation timing.

(A) Experimental approach. One blastomere of a two-cell stage X. laevis embryo was co-microinjected with mRNA to alter nuclear import factor levels and fluorescently labeled dextran as a cell tracer. Embryos were allowed to develop to different stages to assess effects on developmental progression. (B) Microinjections were performed as shown in (A) with 250 pg GFP mRNA, 175 pg NTF mRNA, or 250 pg importin α mRNA + 250 pg GFP-LB3 mRNA. These amounts, that maximally affect nuclear size [13, 24], were used in all experiments. Microinjected two-cell embryos were allowed to develop to 13 hpf gastrula. Representative vegetal pole images are shown. Blastopore area was measured and averaged for 7–13 embryos per condition. Blastopore closure appeared symmetric in all conditions. Error bars represent SD. *** p<0.005, * p<0.05.

Fig 2. Differential levels of nuclear import factors in the two halves of an early embryo lead to neural plate curvature.

(A) Two-cell embryos were microinjected as indicated and allowed to develop to 22 hpf neurula. Representative images are shown. (B) Neurula were scored as having normal or curved neural plates by drawing a line through the middle of the embryo. Embryo numbers: n = 19 for GFP, n = 21 for NTF2, n = 7 for imp α + GFP-LB3/NTF2.

Fig 3. Differential levels of nuclear import factors in the two halves of an early embryo lead to bent tadpoles.

(A) Two-cell embryos were microinjected as indicated and allowed to develop into 9 dpf swimming tadpoles. Representative images are shown. Single-headed arrows indicate small eyes. Double-headed arrows indicate bent bodies. (B) Tadpoles were scored as indicated by measuring eye areas and body axis angles. Embryo numbers: n = 10 for GFP, n = 25 for NTF2, n = 18 for imp α + GFP-LB3/NTF2.

Fig 4. Differential levels of nuclear import factors in the two halves of an early embryo lead to smaller froglets.

Two-cell embryos were microinjected as indicated and allowed to develop into 4-month-old froglets. Froglet numbers: trial 1 GFP n = 5, trial 1 NTF2 n = 24, trial 2 GFP n = 28, trial 2 NTF n = 26. (A) Representative froglets. (B) Quantification of froglet length is shown. Average body mass for froglets derived from NTF2-microinjected embryos was 59% ± 11% (average ± SD) of control froglets. Error bars represent SD. *** p<0.005. (C) Scoring of froglets with altered body morphology as indicated. We did not note any obvious differences in the timing of the onset of metamorphosis for GFP- and NTF2-injected animals.