Reversal of anchorage-independent multicellular spheroid into a monolayer mimics a metastatic model.

Lack of an in vitro model of metastasis has been a major impediment in understanding the molecular regulation of metastatic processes, and identification of specific therapeutic targets. We have established an in vitro model which displayed the signatures of metastatic phenotype such as migration, invasiveness, chemoresistance and expression of cancer stem-cell markers. This in vitro model was developed by the induction of reversal of multicellular spheroids that were generated by anchorage-independent growth. In vivo data further validated the metastatic phenotype of the in vitro model. Besides delineating the molecular events of metastasis, this model could also improve the screening efficiency of antimetastatic agents.

Mitotic chromosome size scaling in Xenopus.

As rapid divisions without growth generate progressively smaller cells within an embryo, mitotic chromosomes must also decrease in size to permit their proper segregation, but this scaling phenomenon is poorly understood. We demonstrated previously that nuclear and spindle size scale between egg extracts of the related frog species Xenopus tropicalis and Xenopus laevis, but show here that dimensions of isolated mitotic sperm chromosomes do not differ. This is consistent with the hypothesis that chromosome scaling does not occur in early embryonic development when cell and spindles sizes are large and anaphase B segregates chromosomes long distances. To recapitulate chromosome scaling during development, we combined nuclei isolated from different stage Xenopus laevis embryos with metaphase-arrested egg extracts. Mitotic chromosomes derived from nuclei of cleaving embryos through the blastula stage were similar in size to replicated sperm chromosomes, but decreased in area approximately 50% by the neurula stage, reproducing the trend in size changes observed in fixed embryos. Allowing G2 nuclei to swell in interphase prior to mitotic condensation did not increase mitotic chromosome size, but progression through a full cell cycle in egg extract did, suggesting that epigenetic mechanisms determining chromosome size can be altered during DNA replication. Comparison of different sized mitotic chromosomes assembled in vitro provides a tractable system to elucidate underlying molecular mechanisms.

A role for central spindle proteins in cilia structure and function.

Cytokinesis and ciliogenesis are fundamental cellular processes that require strict coordination of microtubule organization and directed membrane trafficking. These processes have been intensely studied, but there has been little indication that regulatory machinery might be extensively shared between them. Here, we show that several central spindle/midbody proteins (PRC1, MKLP-1, INCENP, centriolin) also localize in specific patterns at the basal body complex in vertebrate ciliated epithelial cells. Moreover, bioinformatic comparisons of midbody and cilia proteomes reveal a highly significant degree of overlap. Finally, we used temperature-sensitive alleles of PRC1/spd-1 and MKLP-1/zen-4 in C. elegans to assess ciliary functions while bypassing these proteins' early role in cell division. These mutants displayed defects in both cilia function and cilia morphology. Together, these data suggest the conserved reuse of a surprisingly large number of proteins in the cytokinetic apparatus and in cilia.

High-magnification in vivo imaging of Xenopus embryos for cell and developmental biology.

Embryos of the frog Xenopus laevis are an ideal model system for in vivo imaging of dynamic biological processes, from the inner workings of individual cells to the reshaping of tissues during embryogenesis. Their externally developing embryos are more amenable to in vivo analysis than internally developing mammalian embryos, and the large size of the embryos make them particularly suitable for time-lapse analysis of tissue-level morphogenetic events. In addition, individual cells in Xenopus embryos are larger than those in other vertebrate models, making them ideal for imaging cell behavior and subcellular processes (e.g., following the dynamics of fluorescent fusion proteins in living or fixed cells and tissues). Xenopus embryos are amenable to simple manipulations of gene function, including knockdown and misexpression, and the large number of embryos available allows even an inexperienced researcher to perform hundreds of such manipulations per day. Transgenesis is quite effective as well. Finally, because the fate map of Xenopus embryos is stereotypical, simple targeted microinjections can reliably deliver reagents into specific tissues and cell types for gene manipulation or for imaging. Although yolk opacity can hinder deep imaging in intact embryos, almost any cell in the early embryo can be placed into organotypic culture, such that the cells of interest are directly apposed to the cover glass. Furthermore, live imaging techniques can be complemented with immunostaining and in situ hybridization approaches in fixed tissues. This protocol describes methods for labeling and high-magnification time-lapse imaging of cell biological and developmental processes in Xenopus embryos by confocal microscopy.

In vivo imaging reveals a role for Cdc42 in spindle positioning and planar orientation of cell divisions during vertebrate neural tube closure.

Specialization of the cell-division process is a common feature of developing embryos, but most studies on vertebrate cell division have focused on cells dividing in culture. Here, we used in vivo four-dimensional confocal microscopy to explore the role of Cdc42 in governing cell division in the developing neural epithelium of Xenopus laevis. We find that Cdc42 is crucial for stable positioning of the metaphase spindle in these cells, but was not required for spindle positioning in epidermal epithelial cells. We also find that divisions in the Xenopus neural plate are planar oriented, and that rotations of mitotic spindles are essential for establishing this orientation. When Cdc42 is disrupted, spindles over-rotate and the final orientation of divisions is changed. Finally, the planar orientation of cell divisions in this tissue seems to be independent of planar cell polarity (PCP) signaling and does not require normal neural morphogenesis. Our data provide new insights into the coordination of cell division and morphogenesis in epithelial cell sheets and reveal novel, cell-type-specific roles for Cdc42 in spindle positioning and spindle orientation.

Developmental regulation of central spindle assembly and cytokinesis during vertebrate embryogenesis.

Mitosis and cytokinesis not only ensure the proper segregation of genetic information but also contribute importantly to morphogenesis in embryos. Cytokinesis is controlled by the central spindle, a microtubule-based structure containing numerous microtubule motors and microtubule-binding proteins, including PRC1. We show here that central spindle assembly and function differ dramatically between two related populations of epithelial cells in developing vertebrate embryos examined in vivo. Compared to epidermal cells, early neural epithelial cells undergo exaggerated anaphase chromosome separation, rapid furrowing, and a marked reduction of microtubule density in the spindle midzone. Cytokinesis in normal early neural epithelial cells thus resembles that in cultured vertebrate cells experimentally depleted of PRC1. We find that PRC1 mRNA and protein expression is surprisingly dynamic in early vertebrate embryos and that neural-plate cells contain less PRC1 than do epidermal cells. Expression of excess PRC1 ameliorates both the exaggerated anaphase and reduced midzone microtubule density observed in early neural epithelial cells. These PRC1-mediated modifications to the cytokinetic mechanism may be related to the specialization of the midbody in neural cells. These data suggest that PRC1 is a dose-dependent regulator of the central spindle in vertebrate embryos and demonstrate unexpected plasticity to fundamental mechanisms of cell division.

Whole-mount fluorescence immunocytochemistry on Xenopus embryos.

INTRODUCTIONImmunocytochemistry (ICC) is widely exploited in studying mammalian systems, but is underutilized among Xenopus developmental biologists. This stems, in part, from the relatively small number of Xenopus antibodies available for use in research. Common misconceptions about ICC in Xenopus embryos also prevail, discouraging researchers from trying the procedure. However, ICC with Xenopus is simple and effective. This article describes methods for whole-mount ICC in Xenopus embryos. Also included are simple procedures to quench autofluorescence of Xenopus and to remove surface pigment from embryos which may interfere with fluorescence imaging. The methods described here are useful for detecting tissue-specific probes (e.g., 12/101 to detect somites). They are also effective for imaging the cytoskeleton (e.g., α-tubulin to detect microtubules) or localizing specific proteins at the subcellular level (e.g., ZO-1 to detect tight junctions). In addition, combining ICC with in situ hybridization is simple and highly effective.