In vivo high-content imaging and regression analysis reveal non-cell autonomous functions of Shroom3 during neural tube closure.

During neural tube closure, neural ectoderm cells constrict their apical surfaces to bend and fold the tissue into a tube that will become the central nervous system. Recent data from mice and humans with neural tube defects suggest that key genes required for neural tube closure can exert non-cell autonomous effects on cell behavior, but the nature of these effects remains obscure. Here, we coupled tissue-scale, high-resolution time-lapse imaging of the closing neural tube of Xenopus to multivariate regression modeling, and we show that medial actin accumulation drives apical constriction non-autonomously in neighborhoods of cells, rather than solely in individual cells. To further explore this effect, we examined mosaic crispant embryos and identified both autonomous and non-autonomous effects of the apical constriction protein Shroom3.

Global analysis of cell behavior and protein dynamics reveals region-specific roles for Shroom3 and N-cadherin during neural tube closure.

Failures of neural tube closure are common and serious birth defects, yet we have a poor understanding of the interaction of genetics and cell biology during neural tube closure. Additionally, mutations that cause neural tube defects (NTDs) tend to affect anterior or posterior regions of the neural tube but rarely both, indicating a regional specificity to NTD genetics. To better understand the regional specificity of cell behaviors during neural tube closure, we analyzed the dynamic localization of actin and N-cadherin via high-resolution tissue-level time-lapse microscopy during Xenopus neural tube closure. To investigate the regionality of gene function, we generated mosaic mutations in shroom3, a key regulator or neural tube closure. This new analytical approach elucidates several differences between cell behaviors during cranial/anterior and spinal/posterior neural tube closure, provides mechanistic insight into the function of shroom3, and demonstrates the ability of tissue-level imaging and analysis to generate cell biological mechanistic insights into neural tube closure.

Assays for Apical Constriction Using the Xenopus Model.

Apical constriction refers to the active, actomyosin-driven process that reduces apical cell surface area in epithelial cells. Apical constriction is utilized in epithelial morphogenesis during embryonic development in multiple contexts, such as gastrulation, neural tube closure, and organogenesis. Defects in apical constriction can result in congenital birth defects, yet our understanding of the molecular control of apical constriction is relatively limited. To uncover new genetic regulators of apical constriction and gain mechanistic insight into the cell biology of this process, we need reliable assay systems that allow real-time observation and quantification of apical constriction as it occurs and permit gain- and loss-of-function analyses to explore gene function and interaction during apical constriction. In this chapter, we describe using the early Xenopus embryo as an assay system to investigate molecular mechanisms involved in apical constriction during both gastrulation and neurulation.

Unique and redundant ß-catenin regulatory roles of two Dishevelled paralogs during C. elegans asymmetric cell division.

The Wnt/ß-catenin signaling pathway is utilized across metazoans. However, the mechanism of signal transduction, especially dissociation of the ß-catenin destruction complex by Dishevelled proteins, remains controversial. Here, we describe the function of the Dishevelled paralogs DSH-2 and MIG-5 in the Wnt/ß-catenin asymmetry (WßA) pathway in Caenorhabditis elegans, where WßA drives asymmetric cell divisions throughout development. We find that DSH-2 and MIG-5 redundantly regulate cell fate in hypodermal seam cells. Similarly, both DSH-2 and MIG-5 are required for positive regulation of SYS-1 (a C. elegans ß-catenin), but MIG-5 has a stronger effect on the polarity of SYS-1 localization. We show that MIG-5 controls cortical APR-1 (the C. elegans APC) localization. DSH-2 and MIG-5 both regulate the localization of WRM-1 (another C. elegans ß-catenin), acting together as negative regulators of WRM-1 nuclear localization. Finally, we demonstrate that overexpression of DSH-2 or MIG-5 in seam cells leads to stabilization of SYS-1 in the anterior seam daughter, solidifying the Dishevelled proteins as positive regulators of SYS-1. Overall, we have further defined the role of Dishevelled in the WßA signaling pathway, and demonstrated that DSH-2 and MIG-5 regulate cell fate, ß-catenin nuclear levels and the polarity of ß-catenin regulation.

Asymmetric Wnt Pathway Signaling Facilitates Stem Cell-Like Divisions via the Nonreceptor Tyrosine Kinase FRK-1 in Caenorhabditis elegans.

Asymmetric cell division is critical during development, as it influences processes such as cell fate specification and cell migration. We have characterized FRK-1, a homolog of the mammalian Fer nonreceptor tyrosine kinase, and found it to be required for differentiation and maintenance of epithelial cell types, including the stem cell-like seam cells of the hypodermis. A genomic knockout of frk-1, allele ok760, results in severely uncoordinated larvae that arrest at the L1 stage and have an excess number of lateral hypodermal cells that appear to have lost asymmetry in the stem cell-like divisions of the seam cell lineage. frk-1(ok760) mutants show that there are excess lateral hypodermal cells that are abnormally shaped and smaller in size compared to wild type, a defect that could be rescued only in a manner dependent on the kinase activity of FRK-1. Additionally, we observed a significant change in the expression of heterochronic regulators in frk-1(ok760) mutants. However, frk-1(ok760) mutants do not express late, nonseam hypodermal GFP markers, suggesting the seam cells do not precociously differentiate as adult-hyp7 cells. Finally, our data also demonstrate a clear role for FRK-1 in seam cell proliferation, as eliminating FRK-1 during the L3-L4 transition results in supernumerary seam cell nuclei that are dependent on asymmetric Wnt signaling. Specifically, we observe aberrant POP-1 and WRM-1 localization that is dependent on the presence of FRK-1 and APR-1. Overall, our data suggest a requirement for FRK-1 in maintaining the identity and proliferation of seam cells primarily through an interaction with the asymmetric Wnt pathway.

The tumor suppressor APC differentially regulates multiple ß-catenins through the function of axin and CKIα during C. elegans asymmetric stem cell divisions.

The APC tumor suppressor regulates diverse stem cell processes including gene regulation through Wnt-ß-catenin signaling and chromosome stability through microtubule interactions, but how the disparate functions of APC are controlled is not well understood. Acting as part of a Wnt-ß-catenin pathway that controls asymmetric cell division, Caenorhabditis elegans APC, APR-1, promotes asymmetric nuclear export of the ß-catenin WRM-1 by asymmetrically stabilizing microtubules. Wnt function also depends on a second ß-catenin, SYS-1, which binds to the C. elegans TCF POP-1 to activate gene expression. Here, we show that APR-1 regulates SYS-1 levels in asymmetric stem cell division, in addition to its known role in lowering nuclear levels of WRM-1. We demonstrate that SYS-1 is also negatively regulated by the C. elegans homolog of casein kinase 1α (CKIα), KIN-19. We show that KIN-19 restricts APR-1 localization, thereby regulating nuclear WRM-1. Finally, the polarity of APR-1 cortical localization is controlled by PRY-1 (C. elegans Axin), such that PRY-1 controls the polarity of both SYS-1 and WRM-1 asymmetries. We propose a model whereby Wnt signaling, through CKIα, regulates the function of two distinct pools of APC - one APC pool negatively regulates SYS-1, whereas the second pool stabilizes microtubules and promotes WRM-1 nuclear export.

Wide variation in ploidy level and genome size in a New Zealand freshwater snail with coexisting sexual and asexual lineages.

Natural animal populations are rarely screened for ploidy-level variation at a scale that allows detection of potentially important aberrations of common ploidy patterns. This type of screening can be especially important for the many mixed sexual/asexual systems in which sexuals are presumed to be dioecious diploids and asexuals are assumed to be triploid and all-female. For example, elevation of ploidy level above triploidy can be a source of genetic variation and raises the possibility of gene flow among ploidy levels and to asexual lineages. We used flow cytometry and mtDNA sequencing to characterize ploidy level and genome size in Potamopyrgus antipodarum, a New Zealand freshwater snail where obligate sexual (presumed diploid and dioecious) and obligate apomictic asexual (presumed triploid and nearly all female) individuals frequently coexist. We documented the widespread occurrence and multiple origins of polyploid males and individuals with >3× ploidy, and find that both are likely to be descended from asexual females. Our survey also suggested the existence of extensive variation in genome size. The discovery of widespread variation in ploidy level and genome size in such a well-studied system highlights the importance of broad, extensive, and ecologically representative sampling in uncovering ploidy level and genome-size variation in natural populations.