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Marine Biological Laboratory, Woods Hole, Massachusetts 02543
The vertebrate retina develops from a single layer of elongated cells—the optic cup neuroepithelium. At the time of optic cup formation, individual neuroepithelial cells are multipotent and can give rise to any of the cell types found within the differentiated retina (1). As the optic cup neuroepithelium proliferates, the repertoire of cell type fates becomes restricted. Retinal cell specification, the commitment to differentiate as one particular cell type, occurs at or following the final cell division. The underlying mechanisms of cellular specification that generate the diversity of retinal cell types are unknown.
In many invertebrate epithelial cell types, as well as the rat neuroepithelium, the plane of cell division is regulated during development by rapidly reorienting the metaphase chromosomal plane relative to the plane formed by the cellular sheet (2,3). With regard to cellular specification, particular metaphase orientations often correlate with specific cell fates for each daughter cell. Underlying this correlation, studies in both Caenorhabditis elegans and Drosophila melanogaster have demonstrated that the orientation of the mitotic cleavage plane can dictate whether asymmetically distributed mRNAs or proteins are inherited equally or unequally by the two daughter cells (4). Whether vertebrate retinal cells regulate their mitotic cleavage plane though metaphase reorientations is addressed in this study.
To assess metaphase chromosomal plane orientations in a vertebrate retina, newly fertilized zebrafish embryos (1–8 cell stage) were injected with 5 nl of plasmid DNA (0.1 µg/µl) encoding a fusion protein of histone H2B and GFP (H2B::GFP) (5). At this concentration, expression was mosaic. This fusion protein associates with chromosomes throughout the cell cycle in an inert manner, thus fluorescently labeling a subset of the embryo’s cell nuclei. At 22 hours post fertilization (hpf), injected embryos were prepared for time-lapse microscopy. Zebrafish were anesthetized with MS222 (to inhibit spontaneous movements), treated with 0.2 mM 1-phenyl-2-thiourea (to block pigmentation), and embedded in 1.5% agarose (to immobilize the embryo). Labeled proliferating retinal neuroepithelial cells were imaged using a 40x water emersion objective on an upright epifluorescent microscope. Z-series, 50–60 µm in depth, were collected with a cooled CCD camera at intervals of 1–2 min over periods of 10–24 h. At 22 hpf, the retinoblast pool in zebrafish is expanding because all cells of the optic cup neuroepithelium are proliferative with an 8–10 h cell cycle (6).
Mitoses were observed at the apical border of the neuroepithelium adjacent to the retinal pigment epithelium (RPE). Only mitotic cells unobstructed by other labeled cells were scored. A proportion (8/86) of these observable mitoses showed cleavage plane reorientations (Fig. 1). For all cells, the time required to progress from metaphase (initial chromatin condensation) to cell division (end of karyokinesis) showed a range of 9 to 16 min with a mean of 12.8 ± 2.1 min. No significant difference in this time was observed between cells that reoriented their metaphase plate and cells that did not (12.5 ± 2.7 min vs. 12.8 ± 2.1 min). Interestingly, each cell that did rotate spindles shifted its chromosomes by 90 ° so that the plane of cell division was perpendicular to the plane of the neuroepithelial sheet. Cells that did not rotate metaphase chromosomes also cleaved with the axis of separation perpendicular to the RPE-neuroepithelial border.
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The main result of these studies is the demonstration of mitotic cleavage plane reorientations in a vertebrate retina. More generally, by observing mitotic behaviors in situ within a living embryo, cell cycle parameters such as M-phase length or mitotic spindle behavior can be measured directly for individual cells, and heterogeneity can be assessed. This has not been possible with traditional population studies that use cell cycle markers in tissue sections. This experimental system also provides the framework to integrate studies of cleavage plane orientation, asymmetric distribution of mRNA or protein, and cell fate decisions in a single biological context. Lastly, the genetic manipulability of zebrafish will enable mechanistic studies for each of these processes.
This work was funded by generous support from the Grass Foundation. The author also thanks John Dowling, Scott Fraser, and Reinhard Köster for their generosity and advice.
Literature Cited
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