No abstract

Through the injection of f-aequorin (a calcium-specific luminescent reporter) and the use of an imaging photon detector, we see a distinct localized elevation of intracellular calcium that accompanies the appearance of the first furrow arc at the blastodisc surface: the furrow positioning signal. As the leading edges of the arc progress outward toward the margins of the blastodisc, they are accompanied by two subsurface slow calcium waves moving at about 0.2 micron/s: the furrow propagation signal. As these wave fronts approach the edge of the blastodisc, another calcium signal arises in the central region where the positioning signal originally appeared. Like the propagation signal, it extends outward to the margins of the blastodisc, but in this case it also moves downward, accompanying the deepening process that separates the daughter cells: the furrow deepening signal. Both of these furrow deepening progressions move at around 0.1 to 0.2 micron/s. The deepening signal begins to diminish from the center outward, returning to precleavage resting levels on completion of cytokinesis. The signaling sequence is repeated during the second cell division cycle. These localized transients do not require external calcium and they can be dissipated after they have begun by introducing calcium shuttle buffers, resulting in furrow delocalization and regression. They also occur in parthenogenetically activated eggs in which, in an attenuated form, they accompany abortive cleavages.

Formation of female gametes requires acentriolar spindle assembly during meiosis. Mitotic spindles organize from centrosomes and via local activation of the RanGTPase on chromosomes. Vertebrate oocytes present a RanGTP gradient centred on chromatin at all stages of meiotic maturation. However, this gradient is dispensable for assembly of the first meiotic spindle. To understand this meiosis I peculiarity, we studied TPX2, a Ran target, in mouse oocytes. Strikingly, TPX2 activity is controlled at the protein level through its accumulation from meiosis I to II. By RNAi depletion and live imaging, we show that TPX2 is required for spindle assembly via two distinct functions. It controls microtubule assembly and spindle pole integrity via the phosphorylation of TACC3, a regulator of MTOCs activity. We show that meiotic spindle formation in vivo depends on the regulation of at least a target of Ran, TPX2, rather than on the regulation of the RanGTP gradient itself.

The activation process in a variety of deuterostome and protostome eggs is accompanied by cytosolic calcium transients that usually take the form of either a single or multiple propagating waves. Here we report that the eggs of zebrafish (Danio rerio) are no exception in that they generate a single activation wave that traverses the egg at a velocity of around 9 microm/s. There appears, however, to be no difference between the calcium-mediated activation response of eggs with regard to the presence or absence of sperm in the spawning medium. This leads us to suggest that these eggs are normally activated when they come in contact with their spawning medium and are then subsequently fertilized. The aspermic wave is initiated at the animal pole in the region of the micropyle, appears to propagate mainly through the yolk-free egg cortex, and then terminates at the vegetal pole. As neither sperm nor external calcium is required for the initiation (or propagation) of the activation wave, this suggests that an alternative wave trigger must be involved.

2+-release messenger IP 3 , suggesting that brom bones is a regulator of IP 3 -mediated Ca 2+ release at fertilization. Interestingly, brom bones mutant embryos also display defects in dorsoventral axis formation accompanied by a disorganized cortical microtubule network, which is known to be crucial for dorsal axis formation. We provide evidence that the impaired microtubule organization is associated with non-exocytosed cortical granules from the earlier egg activation defect. Positional cloning of the brom bones gene reveals that a premature stop codon in the gene encoding hnRNP I (referred to here as hnrnp I) underlies the abnormalities. Our studies therefore reveal an important new role of hnrnp I in regulating the fundamental process of IP 3 -mediated Ca 2+ release at egg activation.

We report evidence to suggest that during the first few meroblastic cell divisions in zebrafish embryos a dynamic population of central-spindle microtubules serve a crucial function in positioning the cleavage furrow at the surface of the blastoderm. Originating from the mid-zone of the mitotic spindle they develop into what we term a mid-spindle 'pre-furrowing microtubule array' that expands upward and outward from the spindle mid-zone towards the blastodisc surface. We suggest that this structure transmits positional information to the blastodisc cortex that results in the correctly positioned assembly of the cytokinetic contractile apparatus. We also propose that the pre-furrowing microtubule array then develops into a furrow-ingression microtubule array that helps direct and assemble the deepening furrow as it cuts its way through the blastodisc. Due to the location of its origin, the pre-furrowing microtubule array serves to successfully separate the daughter nuclei and thus equally divide the blastoderm. Furthermore, co-localization with elements of the cortical endoplasmic reticulum and their inositol 1,4,5-trisphosphate receptors suggests that the pre-furrowing microtubule array may also play a role in organizing localized Ca 2+ transients that have been shown to be essential to the furrow positioning, propagation and deepening process during cytokinesis in zebrafish embryos.

We report that the first localized Ca(2+) transient visualized in the blastodisc cortex of post-mitotic zebrafish zygotes has unique features. We confirm that this initial 'furrow positioning' Ca(2+) transient precedes the physical appearance of the first cleavage furrow at the blastodisc surface and that it has unique dynamics, which distinguish it from the subsequent furrow propagation transients that develop from it. This initial transient displays a distinct rising phase that peaks prior to the initiation of the two linear, subsurface, self-propagating Ca(2+) waves that constitute the subsequent furrow propagation transient. Through the carefully timed introduction of the Ca(2+) buffer, dibromo-BAPTA, we also demonstrate the absolute requirement of this initial rising phase Ca(2+) transient in positioning the furrow at the blastodisc surface: no rising phase transient, no cleavage furrow. Likewise, the introduction of the inositol 1,4,5-trisphosphate receptor (IP3R) antagonist, 2-aminoethoxydiphenyl borate, eliminates both the rising phase transient and the appearance of the furrow at the cell surface. On the other hand, antagonists of the ryanodine receptor and NAADP-sensitive channels, or simply bathing the zygote in Ca(2+)-free medium, have no effect on the generation of the rising phase positioning transient or the appearance of the furrow at the surface. This suggests that like the subsequent propagation and deepening/zipping Ca(2+) transients, the rising phase furrow positioning transient is also generated specifically by Ca(2+) released via IP3Rs. We propose, however, that despite being generated by a similar Ca(2+) release mechanism, the unique features of this initial transient suggest that it might be a distinct signal with a specific function associated with positioning the cleavage furrow at the blastodisc surface.

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