Paul Skoglund

AN EVALUATION OF TWO-PHOTON EXCITATION VERSUS CONFOCAL AND DIGITAL DECONVOLUTION FLUORESCENCE MICROSCOPY IMAGING IN XENOPUS MORPHOGENESIS

The ability to visualize cell motility occurring deep in the context of opaque tissues will allow many currently intractable issues in developmental biology and organogenesis to be addressed. In this study, we compare two‐photon excitation with laser scanning confocal and conventional digital deconvolution fluorescence microscopy, using the same optical configuration, for their ability to resolve cell shape deep in Xenopus gastrula and neurula tissues. The two‐photon microscope offers better depth penetration and less autofluorescence compared to confocal and conventional deconvolution imaging. Both two‐photon excitation and confocal microscopy also provide improved rejection of “out‐of‐focus” noise and better lateral and axial resolution than conventional digital deconvolution microscopy. Deep Xenopus cells are best resolved by applying the digital deconvolution method on the two‐photon images. We have also found that the two‐photon has better depth penetration without any degradation in the image quality of interior sections compared to the other two techniques. Also, we have demonstrated that the quality of the image changes at different depths for various excitation powers. Microsc. Res. Tech. 47:172–181, 1999.

Integration of planar cell polarity and ECM signaling in elongation of the vertebrate body plan

The shaping of the vertebrate embryonic body plan depends heavily on the narrowing and lengthening (convergence and extension) of embryonic tissues by cell intercalation, a process by which cells actively crawl between one another along the axis of convergence to produce a narrower, longer array. We discuss recent evidence that the vertebrate non-canonical Wnt/Planar Cell Polarity (PCP) pathway, known to directly function in polarizing the movements of intercalating cells, is also involved in the localized assembly of extracellular matrix (ECM). These cell-ECM interactions, in turn, are necessary for expression of the oriented, polarized cell intercalation. The mechanism of PCP/ECM interactions, their molecular signaling, and their mechanical consequences for morphogenesis are discussed with the goal of identifying important unsolved issues.

Xenopus fibrillin is expressed in the organizer and is the earliest component of matrix at the developing notochord‐somite boundary

We identify a Xenopus fibrillin homolog (XF), and show that its earliest developmental expression is in presumptive dorsal mesoderm at gastrulation, and that XF expression is regulated by mesoderm‐inducing factors in animal cap assays. XF protein is also first detected in presumptive mesoderm, but is concentrated specifically into extracellular‐matrix structures that begin to develop de novo by mid‐gastrulation at both of the bilateral presumptive notochord‐somite boundaries. Later in embryogenesis, XF protein is localized to the extracellular matrix at tissue boundaries, where it is found surrounding the notochord, the somites, and the neural tube, as well as under the epidermis. This pattern of protein deposition combines to give the appearance of an “embryonic skeleton,” suggesting that one role for XF is to serve as a mechanical element in the embryo prior to bone deposition. Developmental Dynamics 235:1974–1983, 2006.

Molecular model for force production and transmission during vertebrate gastrulation.

Vertebrate embryos undergo dramatic shape changes at gastrulation that require locally produced and anisotropically applied forces, yet how these forces are produced and transmitted across tissues remains unclear. We show that depletion of myosin regulatory light chain (RLC) levels in the embryo blocks force generation at gastrulation through two distinct mechanisms: destabilizing the myosin II (MII) hexameric complex and inhibiting MII contractility. Molecular dissection of these two mechanisms demonstrates that normal convergence force generation requires MII contractility and we identify a set of molecular phenotypes correlated with both this failure of convergence force generation in explants and of blastopore closure in whole embryos. These include reduced rates of actin movement, alterations in C-cadherin dynamics and a reduction in the number of polarized lamellipodia on intercalating cells. By examining the spatial relationship between C-cadherin and actomyosin we also find evidence for formation of transcellular linear arrays incorporating these proteins that could transmit mediolaterally oriented tensional forces. These data combine to suggest a multistep model to explain how cell intercalation can occur against a force gradient to generate axial extension forces. First, polarized lamellipodia extend mediolaterally and make new C-cadherin-based contacts with neighboring mesodermal cell bodies. Second, lamellipodial flow of actin coalesces into a tension-bearing, MII-contractility-dependent node-and-cable actin network in the cell body cortex. And third, this actomyosin network contracts to generate mediolateral convergence forces in the context of these transcellular arrays. Summary: The analysis of actomyosin and C-cadherin dynamics in Xenopus embryos suggests a multistep model to explain how cell intercalation can occur against a force gradient to drive axial extension.

Kif2a depletion generates chromosome segregation and pole coalescence defects in animal caps and inhibits gastrulation of the Xenopus embryo

Characterization of Kif2a in Xenopus embryos identifies new roles for the Kif2a microtubule depolymerase in coordinating cytokinesis and centrosome coalescence. In addition, defects in mitosis can inhibit large-scale developmental movements in vertebrate tissues.

Characterization of convergent thickening, a major convergence force producing morphogenic movement in amphibians

We characterize the morphogenic process of convergent thickening (CT), which occurs in the involuting marginal zone (IMZ) during gastrulation of Xenopus, the African clawed frog. CT was described previously as the tendency of explants of the ventral IMZ of Xenopus to converge their circumblastoporal dimension and thicken their radial dimension (Keller and Danilchik 1988). Here we show that CT occurs from the onset of gastrulation, initially throughout the pre-involution IMZ. We suggest that CT is driven by an increase in the interfacial tension between the deep IMZ and its epithelium, resulting in cells of the deep IMZ tending to minimize their surface area. In explants, this results in a progressive shortening (convergence) of the IMZ along its longer mediolateral axis and thickening in the orthogonal planes, and can generate tensile force (Shook et al. 2018). In vivo, convergence of the annular IMZ generates circumferential tension, closing the blastopore. These results provide the first clear example of a tensile morphogenic force from a Holtfreterian/Steinbergian change in tissue affinity.

Identification of drivers of aneuploidy in breast tumors

SUMMARY Although aneuploidy is found in the majority of tumors, the degree of aneuploidy varies widely. It is unclear how cancer cells become aneuploid or how highly aneuploid tumors are different from those of more normal ploidy. We developed a simple computational method that measures the degree of aneuploidy or structural rearrangements of large chromosome regions of 522 human breast tumors from The Cancer Genome Atlas (TCGA). Highly aneuploid tumors overexpress activators of mitotic transcription and the genes encoding proteins that segregate chromosomes. Overexpression of three mitotic transcriptional regulators, E2F1, MYBL2, and FOXM1, is sufficient to increase the rate of lagging anaphase chromosomes in a non-transformed vertebrate tissue, demonstrating that this event can initiate aneuploidy. Highly aneuploid human breast tumors are also enriched in TP53 mutations. TP53 mutations co-associate with the overexpression of mitotic transcriptional activators, suggesting that these events work together to provide fitness to breast tumors.