Characterization of mid-spindle microtubules during furrow positioning in early cleavage period zebrafish embryos

Abstract

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.

Calcium signaling during the early meroblastic cleavages of zebrafish and medaka embryos

“…Two of these, however, are addressed by Guo et al: The first is the linear (rather than radial) nature of the propagating cytokinetic Ca 2+ waves, while the second is the relatively long duration of the cytokinetic Ca 2+ signals themselves. In the case of the former, Guo et al confirm earlier reports (Lee et al, 2003;Lee et al, 2004) that during cytokinesis the endoplasmic reticulum (ER) in the blastomeres of cleavage period zebrafish embryos is rearranged into linear arrays that reflect the plane of the cleavage furrow. As a result, any inositol 1,4,5-trisphosphate receptor (IP 3 R)-mediated Ca 2+ transient will propagate as a linear Ca 2+ wave along the plane of the furrow, rather than a radial wave as seen during activation/fertilization of many eggs, including zebrafish (Lee et al, 1999;Stricker, 1999).…”

Calcium signalling during the cleavage period of zebrafish development

“…Preliminary evidence is now beginning to accumulate to suggest what the functional role of each of these transients might be, although the precise molecular details of the interactions between localized Ca 2C elevations and Ca 2C -sensitive cytoskeletal and cytosolic signalling elements still remain unclear. A number of hypothetical models have been proposed that attempt to link these Ca 2C transients to specific cytokinetic events ( Fluck et al 1991;Webb et al 1997;Lee et al 2004Lee et al , 2006. We have attempted to summarize and update these models in figure 2.…”

“…Furthermore, we also started to explore the upstream events that might be involved in generating the furrow positioning Ca 2C transient and presented evidence to demonstrate that a dynamic array of microtubules may be involved. We showed that this array, which we termed the 'pre-furrowing microtubule array' (or pf-MTA), originates from the mid-zone of the mitotic spindle and then expands both upward and outward towards the surface of the blastodisc (Lee et al 2004). In addition, we showed that this dynamic pf-MTA eventually colocalizes with a zone of corticular ER (and associated IP 3 Rs) in the blastoderm cortex just before the morphological appearance of the cleavage furrow at the blastodisc surface and suggested that the array might be involved in reorganizing the ER (and thus the IP 3 Rs) required to generate the Ca 2C signals that are essential for cleavage furrow formation in zebrafish embryos (Lee et al 2004).…”

“…We showed that this array, which we termed the 'pre-furrowing microtubule array' (or pf-MTA), originates from the mid-zone of the mitotic spindle and then expands both upward and outward towards the surface of the blastodisc (Lee et al 2004). In addition, we showed that this dynamic pf-MTA eventually colocalizes with a zone of corticular ER (and associated IP 3 Rs) in the blastoderm cortex just before the morphological appearance of the cleavage furrow at the blastodisc surface and suggested that the array might be involved in reorganizing the ER (and thus the IP 3 Rs) required to generate the Ca 2C signals that are essential for cleavage furrow formation in zebrafish embryos (Lee et al 2004). The reorganized ER assumes a linear architecture and this may explain the generation of linear rather than radial Ca 2C waves (Fluck et al 1991).…”

Requirement for a localized, IP3R-generated Ca2+transient during the furrow positioning process in zebrafish zygotes

“…From our previous imaging studies (Webb et al, 1997;Lee et al, 2004), we determined that the furrow positioning transient lasts for approximately 30-60 s before it develops into the furrow propagation transient. In order to challenge furrow positioning, embryos were removed for either microinjection or medium replacement shortly before or immediately after the furrow positioning transient was observed (i.e.…”