Nuclear Size Scaling during Xenopus Early Development Contributes to Midblastula Transition Timing

Abstract

Summary Early Xenopus laevis embryogenesis is a robust system for investigating mechanisms of developmental timing. After a series of rapid cell divisions with concomitant reductions in cell size, the first major developmental transition is the midblastula transition (MBT), when zygotic transcription begins and cell cycles elongate [1-3]. While the maintenance of a constant nuclear-to-cytoplasmic (N/C) volume ratio is a conserved cellular property [4-7], it has long been recognized that the N/C volume ratio changes dramatically during early Xenopus development [8]. We investigated how changes in nuclear size and the N/C volume ratio during early development contribute to the regulation of MBT timing. While previous studies suggested a role for the N/C volume ratio in MBT timing [1, 9-13], none directly tested the effects of altering nuclear size. In this study, we first quantify blastomere and nuclear sizes in X. laevis embryos, demonstrating that the N/C volume ratio increases prior to the MBT. We then manipulate nuclear volume in embryos by microinjecting different nuclear scaling factors, including import proteins, lamins, and reticulons. Using this approach, we show that increasing the N/C volume ratio in pre-MBT embryos leads to premature activation of zygotic gene transcription and early onset of longer cell cycles. Conversely, decreasing the N/C volume ratio delays zygotic transcription and leads to additional rapid cell divisions. While the DNA-to-cytoplasmic ratio has been implicated in MBT timing [1, 9-18], our data show that nuclear size also contributes to the regulation of MBT timing, demonstrating the functional significance of nuclear size during development.

Recent advances in understanding nuclear size and shape

“…86 NE formation was inhibited in Xenopus egg extract supplemented with a neutralizing Rtn4 antibody, 77 and ectopic Rtn4 expression in early Xenopus embryos led to altered nuclear size. 71 Furthermore, ectopic addition of recombinant Rtn4b to Xenopus egg extract decreased the rate of nuclear expansion, leading to an »2.4-fold reduction in nuclear cross-sectional area (Fig. 1B).…”

“…70 Nuclear size decreases throughout early embryonic development in both X. laevis and X. tropicalis. 67,71,72 Nuclear size reductions prior to the MBT correlate with reduced bulk import rates and levels of cytoplasmic importin a. Ectopic importin a expression was sufficient to increase nuclear size in pre-MBT embryos, while expression of both importin a and lamin B3 was required to increase nuclear size in later developmental stages. 67,71 Experiments in C. elegans similarly demonstrated the involvement of nuclear transport and lamins in nuclear size regulation.…”

“…67,71,72 Nuclear size reductions prior to the MBT correlate with reduced bulk import rates and levels of cytoplasmic importin a. Ectopic importin a expression was sufficient to increase nuclear size in pre-MBT embryos, while expression of both importin a and lamin B3 was required to increase nuclear size in later developmental stages. 67,71 Experiments in C. elegans similarly demonstrated the involvement of nuclear transport and lamins in nuclear size regulation. 73,74 In pre-MBT Xenopus embryos, nuclei expand throughout interphase, while at the MBT and beyond nuclei reach a steady-state size that is smaller than nuclei in pre-MBT embryos.…”

“…A 3-fold decrease in NE surface area occurs between the MBT and gastrulation (stages 10.5-12). 67,71 When large nuclei assembled in…”

“…Rtn4a overexpression has been observed in malignant brain tumor cells, and sufficiently high Rtn expression levels could have a dominant negative effect leading to increased nuclear size, as observed in Xenopus. 71 Downregulation of Rtn4 Interacting Protein 1 (Rtn4IP1) has been observed in thyroid cancers, 98,99 potentially decreasing the efficiency with which Rtn4 shapes tubulated ER and leading to an increase in nuclear size. 100 As another example, Rtn1 is upregulated in malignant pancreatic carcinoma, diffusely infiltrating gliomas, and neuroendocrine tumors, 98,[101][102][103] again possibly influencing ER and nuclear morphology in these cancers.…”