Leveraging Xenopus frog species to study architectural diversity of the mitotic spindle and mechanisms underlying hybrid inviability

Cell division is crucial to life. Across all eukaryotes, this dramatic and complex process relies on the mitotic spindle to segregate chromosomes faithfully into new daughter cells, ensuring the health and survival of the next generation. Despite its conserved components and universal function of transmitting a complete genome, the cell division machinery, including the central mitotic spindle, varies widely across the tree of life in size and morphology. Interestingly, chromosome segregation defects, genome elimination, and embryonic lethality are frequently observed upon hybridization of closely related species. However, whether and how divergent cell division machinery leads to functional defects is unknown. This dissertation is comprised of four projects that span comparative evolutionary, cell, and developmental biology to examine the basis and consequences of divergent cell division machineries by using X. laevis, X. tropicalis, and X. borealis frogs. I develop the use of X. borealis to understand divergence in spindle assembly mechanisms and examine how spindle architecture varies even among closely related frog species. I adapt expansion microscopy for Xenopus extract systems, combining an unparalleled in vitro physiological reconstitution system with high-resolution imaging to measure and dissect microtubule organization to better define spindle morphometrics. We leverage the full power of the Xenopus systems to establish the most tractable vertebrate model for investigating the cellular and molecular mechanisms underlying hybridization outcomes, incompatibility, and genome elimination. Altogether, this work provides important molecular insight and understanding to mechanisms that contribute to spindle assembly and scaling, hybrid inviability, and speciation

Spindle assembly in egg extracts of the Marsabit clawed frog, Xenopus borealis

International audienceEgg extracts of the African clawed frog Xenopus laevis have provided a cell-free system instrumental in elucidating events of the cell cycle, including mechanisms of spindle assembly. Comparison with extracts from the diploid Western clawed frog, Xenopus tropicalis, which is smaller at the organism, cellular and subcellular levels, has enabled the identification of spindle size scaling factors. We set out to characterize the Marsabit clawed frog, Xenopus borealis, which is intermediate in size between the two species, but more recently diverged in evolution from X. laevis than X. tropicalis. X. borealis eggs were slightly smaller than those of X. laevis, and slightly smaller spindles were assembled in egg extracts. Interestingly, microtubule distribution across the length of the X. borealis spindles differed from both X. laevis and X. tropicalis. Extract mixing experiments revealed common scaling phenomena among Xenopus species, while characterization of spindle factors katanin, TPX2, and Ran indicate that X. borealis spindles possess both X. laevis and X. tropicalis features. Thus, X. borealis egg extract provides a third in vitro system to investigate interspecies scaling and spindle morphometric variation

Spindle assembly in egg extracts of the Marsabit clawed frog, Xenopus borealis.

Egg extracts of the African clawed frog Xenopus laevis have provided a cell-free system instrumental in elucidating events of the cell cycle, including mechanisms of spindle assembly. Comparison with extracts from the diploid Western clawed frog, Xenopus tropicalis, which is smaller at the organism, cellular and subcellular levels, has enabled the identification of spindle size scaling factors. We set out to characterize the Marsabit clawed frog, Xenopus borealis, which is intermediate in size between the two species, but more recently diverged in evolution from X. laevis than X. tropicalis. X. borealis eggs were slightly smaller than those of X. laevis, and slightly smaller spindles were assembled in egg extracts. Interestingly, microtubule distribution across the length of the X. borealis spindles differed from both X. laevis and X. tropicalis. Extract mixing experiments revealed common scaling phenomena among Xenopus species, while characterization of spindle factors katanin, TPX2, and Ran indicate that X. borealis spindles possess both X. laevis and X. tropicalis features. Thus, X. borealis egg extract provides a third in vitro system to investigate interspecies scaling and spindle morphometric variation

Preparation of Xenopus borealis and Xenopus tropicalis Egg Extracts for Comparative Cell Biology and Evolutionary Studies

International audienceCytoplasmic extracts prepared from eggs of the African clawed frog Xenopus laevis are extensively used to study various cellular events including the cell cycle, cytoskeleton dynamics, and cytoplasm organization, as well as the biology of membranous organelles and phase-separated non-membrane-bound structures. Recent development of extracts from eggs of other Xenopus allows interspecies comparisons that provide new insights into morphological and biological size variations and underlying mechanisms across evolution. Here, we describe methods to prepare cytoplasmic extracts from eggs of the allotetraploid Marsabit clawed frog, Xenopus borealis, and the diploid Western clawed frog, Xenopus tropicalis. We detail mixing and "hybrid" experiments that take advantage of the physiological but highly accessible nature of extracts to reveal the evolutionary relationships across species. These new developments create a robust and versatile toolbox to elucidate molecular, cell biological, and evolutionary questions in essential cellular processes

Cross-modal size-contrast illusion : acoustic increases in intensity and bandwidth modulate haptic representation of object size

Changes in the retinal size of stationary objects provide a cue to the observer's motion in the environment: Increases indicate the observer's forward motion, and decreases backward motion. In this study, a series of images each comprising a pair of pine-tree figures were translated into auditory modality using sensory substitution software. Resulting auditory stimuli were presented in an ascending sequence (i.e. increasing in intensity and bandwidth compatible with forward motion), descending sequence (i.e. decreasing in intensity and bandwidth compatible with backward motion), or in a scrambled order. During the presentation of stimuli, blindfolded participants estimated the lengths of wooden sticks by haptics. Results showed that those exposed to the stimuli compatible with forward motion underestimated the lengths of the sticks. This consistent underestimation may share some aspects with visual size-contrast effects such as the Ebbinghaus illusion. In contrast, participants in the other two conditions did not show such magnitude of error in size estimation; which is consistent with the "adaptive perceptual bias" towards acoustic increases in intensity and bandwidth. In sum, we report a novel cross-modal size-contrast illusion, which reveals that auditory motion cues compatible with listeners' forward motion modulate haptic representations of object size.Published versio

Molecular conflicts disrupting centromere maintenance contribute to Xenopus hybrid inviability

Although central to evolution, the causes of hybrid inviability that drive reproductive isolation are poorly understood. Embryonic lethality occurs when the eggs of the frog X. tropicalis are fertilized with either X. laevis or X. borealis sperm. We observed that distinct subsets of paternal chromosomes failed to assemble functional centromeres, causing their mis-segregation during embryonic cell divisions. Core centromere DNA sequence analysis revealed little conservation among the three species, indicating that epigenetic mechanisms that normally operate to maintain centromere integrity are disrupted on specific paternal chromosomes in hybrids. In vitro reactions combining X. tropicalis egg extract with either X. laevis or X. borealis sperm chromosomes revealed that paternally matched or overexpressed centromeric histone CENP-A and its chaperone HJURP could rescue centromere assembly on affected chromosomes in interphase nuclei. However, although the X. laevis chromosomes maintained centromeric CENP-A in metaphase, X. borealis chromosomes did not and also displayed ultra-thin regions containing ribosomal DNA. Both centromere assembly and morphology of X. borealis mitotic chromosomes could be rescued by inhibiting RNA polymerase I or preventing the collapse of stalled DNA replication forks. These results indicate that specific paternal centromeres are inactivated in hybrids due to the disruption of associated chromatin regions that interfere with CENP-A incorporation, at least in some cases due to conflicts between replication and transcription machineries. Thus, our findings highlight the dynamic nature of centromere maintenance and its susceptibility to disruption in vertebrate interspecies hybrids

Dynamic redox balance directs the oocyte-to-embryo transition via developmentally controlled reactive cysteine changes

The metabolic and redox state changes during the transition from an arrested oocyte to a totipotent embryo remain uncharacterized. Here, we applied state-of-the-art, integrated methodologies to dissect these changes in Drosophila. We demonstrate that early embryos have a more oxidized state than mature oocytes. We identified specific alterations in reactive cysteines at a proteome-wide scale as a result of this metabolic and developmental transition. Consistent with a requirement for redox change, we demonstrate a role for the ovary-specific thioredoxin Deadhead (DHD). dhd-mutant oocytes are prematurely oxidized and exhibit meiotic defects. Epistatic analyses with redox regulators link dhd function to the distinctive redox-state balance set at the oocyte-to-embryo transition. Crucially, global thiol-redox profiling identified proteins whose cysteines became differentially modified in the absence of DHD. We validated these potential DHD substrates by recovering DHD-interaction partners using multiple approaches. One such target, NO66, is a conserved protein that genetically interacts with DHD, revealing parallel functions. As redox changes also have been observed in mammalian oocytes, we hypothesize a link between developmental control of this cell-cycle transition and regulation by metabolic cues. This link likely operates both by general redox state and by changes in the redox state of specific proteins. The redox proteome defined here is a valuable resource for future investigation of the mechanisms of redox-modulated control at the oocyte-to-embryo transition