Hitoshi Sawa

Components of the transcriptional Mediator complex are required for asymmetric cell division in C. elegans

Asymmetric cell division is a fundamental process that produces cellular diversity during development. In C. elegans, the Wnt signaling pathway regulates the asymmetric divisions of a number of cells including the T blast cell. We found that the let-19 and dpy-22 mutants have defects in their T-cell lineage, and lineage analyses showed that the defects were caused by disruption in the asymmetry of the T-cell division. We found that let-19 and dpy-22 encode homologs of the human proteins MED13/TRAP240 and MED12/TRAP230, respectively, which are components of the Mediator complex. Mediator is a multi-component complex that can regulate transcription by transducing the signals between activators and RNA polymerase in vitro. We also showed that LET-19 and DPY-22 form a complex in vivo with other components of Mediator, SUR-2/MED23 and LET-425/MED6. In the let-19 and dpy-22 mutants, tlp-1, which is normally expressed asymmetrically between the T-cell daughters through the function of the Wnt pathway, was expressed symmetrically in both daughter cells. Furthermore, we found that the let-19 and dpy-22 mutants were defective in the fusion of the Pn.p cell, a process that is regulated by bar-1/β-catenin. Ectopic cell fusion in bar-1 mutants was suppressed by the let-19 or dpy-22 mutations, while defective cell fusion in let-19 mutants was suppressed by lin-39/Hox mutations, suggesting that let-19 and dpy-22 repress the transcription of lin-39. These results suggest that LET-19 and DPY-22 in the Mediator complex repress the transcription of Wnt target genes.

Complex Network of Wnt Signaling Regulates Neuronal Migrations During Caenorhabditis elegans Development

Members of the Wnt family of secreted glycoproteins regulate many developmental processes, including cell migration. We and others have previously shown that the Wnts egl-20, cwn-1, and cwn-2 are required for cell migration and axon guidance. However, the roles in cell migration of all of the Caenorhabditis elegans Wnt genes and their candidate receptors have not been explored fully. We have extended our analysis to include all C. elegans Wnts and six candidate Wnt receptors: four Frizzleds, the sole Ryk family receptor LIN-18, and the Ror receptor tyrosine kinase CAM-1. We show that three of the Wnts, CWN-1, CWN-2, and EGL-20, play major roles in directing cell migrations and that all five Wnts direct specific cell migrations either by acting redundantly or by antagonizing each others function. We report that all four Frizzleds function to direct Q-descendant cell migrations, but only a subset of the putative Wnt receptors function in directing migrations of other cells. Finally, we find striking differences between the phenotypes of the Wnt quintuple and Frizzled quadruple mutants.

Wnt Regulates Spindle Asymmetry to Generate Asymmetric Nuclear β-Catenin in C. elegans

Extrinsic signals received by a cell can induce remodeling of the cytoskeleton, but the downstream effects of cytoskeletal changes on gene expression have not been well studied. Here, we show that during telophase of an asymmetric division in C. elegans, extrinsic Wnt signaling modulates spindle structures through APR-1/APC, which in turn promotes asymmetrical nuclear localization of WRM-1/β-catenin and POP-1/TCF. APR-1 that localized asymmetrically along the cortex established asymmetric distribution of astral microtubules, with more microtubules found on the anterior side. Perturbation of the Wnt signaling pathway altered this microtubule asymmetry and led to changes in nuclear WRM-1 asymmetry, gene expression, and cell-fate determination. Direct manipulation of spindle asymmetry by laser irradiation altered the asymmetric distribution of nuclear WRM-1. Moreover, laser manipulation of the spindles rescued defects in nuclear POP-1 asymmetry in wnt mutants. Our results reveal a mechanism in which the nuclear localization of proteins is regulated through the modulation of microtubules.

β‐Catenin asymmetry is regulated by PLA1 and retrograde traffic in C. elegans stem cell divisions

Asymmetric division is an important property of stem cells. In Caenorhabditis elegans, the Wnt/β‐catenin asymmetry pathway determines the polarity of most asymmetric divisions. The Wnt signalling components such as β‐catenin localize asymmetrically to the cortex of mother cells to produce two distinct daughter cells. However, the molecular mechanism to polarize them remains to be elucidated. Here, we demonstrate that intracellular phospholipase A1 (PLA1), a poorly characterized lipid‐metabolizing enzyme, controls the subcellular localizations of β‐catenin in the terminal asymmetric divisions of epithelial stem cells (seam cells). In mutants of ipla‐1, a single C. elegans PLA1 gene, cortical β‐catenin is delocalized and the asymmetry of cell‐fate specification is disrupted in the asymmetric divisions. ipla‐1 mutant phenotypes are rescued by expression of ipla‐1 in seam cells in a catalytic activity‐dependent manner. Furthermore, our genetic screen utilizing ipla‐1 mutants reveals that reduction of endosome‐to‐Golgi retrograde transport in seam cells restores normal subcellular localization of β‐catenin to ipla‐1 mutants. We propose that membrane trafficking regulated by ipla‐1 provides a mechanism to control the cortical asymmetry of β‐catenin.

Wnt signaling in C. elegans

Wnt proteins are secreted lipid-modified glycoproteins that control many aspects of development in organisms ranging from sponges to vertebrates. Wnt proteins are also important regulators of C. elegans development, with functions in processes as diverse as cell fate specification, asymmetric cell division, cell migration and synapse formation. In this review, we will give an overview of what we currently know about the signaling mechanisms that mediate these different functions of Wnt.

Critical role of Caenorhabditis elegans homologs of Cds1 (Chk2)-related kinases in meiotic recombination

ABSTRACT Although chromosomal segregation at meiosis I is the critical process for genetic reassortment and inheritance, little is known about molecules involved in this process in metazoa. Here we show by utilizing double-stranded RNA (dsRNA)-mediated genetic interference that novel protein kinases (Ce-CDS-1 and Ce-CDS-2) related to Cds1 (Chk2) play an essential role in meiotic recombination inCaenorhabditis elegans. Injection of dsRNA into adult animals resulted in the inhibition of meiotic crossing over and induced the loss of chiasmata at diakinesis in oocytes of F1animals. However, electron microscopic analysis revealed that synaptonemal complex formation in pachytene nuclei of the same progeny of injected animals appeared to be normal. Thus, Ce-CDS-1 and Ce-CDS-2 are the first example of Cds1-related kinases that are required for meiotic recombination in multicellular organisms.

Extracellular control of PAR protein localization during asymmetric cell division in the C. elegans embryo

The axis of asymmetric cell division is controlled to determine the future position of differentiated cells during animal development. The asymmetric localization of PAR proteins in the Drosophila neuroblast and C. elegans embryo are aligned with the axes of the embryo. However, whether extracellular or intracellular signals determine the orientation of the localization of PAR proteins remains controversial. In C. elegans, the P0 zygote and germline cells (P1, P2, and P3) undergo a series of asymmetric cell divisions. Interestingly, the axis of the P0 and P1 divisions is opposite to that of the P2 and P3 divisions. PAR-2, a ring-finger protein, and PAR-1, a kinase, relocalize to the anterior side of the P2 and P3 germline precursors at the site of contact with endodermal precursors. Using an in vitro method, we have found that the PAR-2 protein is distributed asymmetrically in the absence of extracellular signals, but the orientation of the protein localization in the P2 and P3 cells is determined by contact with endodermal precursor cells. Our mutant analyses suggest that mes-1 and src-1, which respectively encode a transmembrane protein and a tyrosine kinase, were not required to establish the asymmetric distribution of PAR-2, but were required to determine its orientation at the site of contact with the endodermal precursors. The PAR-2 localization during the asymmetric P2 and P3 divisions is controlled by extracellular signals via MES-1/SRC-1 signaling. Our findings suggest that Src functions as an evolutionarily conserved molecular link that coordinates extrinsic cues with PAR protein localization.

RMD-1, a novel microtubule-associated protein, functions in chromosome segregation in Caenorhabditis elegans

For proper chromosome segregation, the sister kinetochores must attach to microtubules extending from the opposite spindle poles. Any errors in microtubule attachment can induce aneuploidy. In this study, we identify a novel conserved Caenorhabditis elegans microtubule-associated protein, regulator of microtubule dynamics 1 (RMD-1), that localizes to spindle microtubules and spindle poles. Depletion of RMD-1 induces severe defects in chromosome segregation, probably through merotelic attachments between microtubules and chromosomes. Although rmd-1 embryos also have a mild defect in microtubule growth, we find that mutants of the microtubule growth regulator XMAP215/ZYG-9 show much weaker segregation defects. This suggests that the microtubule growth defect in rmd-1 embryos does not cause abnormal chromosome segregation. We also see that RMD-1 interacts with aurora B in vitro. Our results suggest that RMD-1 functions in chromosome segregation in C. elegans embryos, possibly through the aurora B–mediated pathway. Human homologues of RMD-1 could also bind microtubules, which would suggest a function for these proteins in chromosome segregation during mitosis in other organisms as well.

Double bromodomain protein BET-1 and MYST HATs establish and maintain stable cell fates in C. elegans

The maintenance of cell fate is important for normal development and tissue homeostasis. Epigenetic mechanisms, including histone modifications, are likely to play crucial roles in cell-fate maintenance. However, in contrast to the established functions of histone methylation, which are mediated by the polycomb proteins, the roles of histone acetylation in cell-fate maintenance are poorly understood. Here, we show that the C. elegans acetylated-histone-binding protein BET-1 is required for the establishment and maintenance of stable fate in various lineages. In most bet-1 mutants, cells adopted the correct fate initially, but at later stages they often transformed into a different cell type. By expressing BET-1 at various times in development and examining the rescue of the Bet-1 phenotype, we showed that BET-1 functions both at the time of fate acquisition, to establish a stable fate, and at later stages, to maintain the established fate. Furthermore, the disruption of the MYST HATs perturbed the subnuclear localization of BET-1 and caused bet-1-like phenotypes, suggesting that BET-1 is recruited to its targets through acetylated histones. Our results therefore indicate that histone acetylation plays a crucial role in cell-fate maintenance.

Specification of neurons through asymmetric cell divisions

The brain requires diverse neuronal subtypes to carry out its complex functions. Many types of neurons are produced through asymmetric division, and the molecular mechanisms of asymmetric division have been extensively studied in C. elegans and Drosophila. In these model organisms, the same molecular mechanisms regulate asymmetric divisions throughout development, although diverse cell types are created. How these common mechanisms for asymmetric division can specify diverse neuronal fates, however, is still being discovered. Recent studies suggest that neurons are specified by the combined effects of asymmetric divisions, which are regulated by common mechanisms, and specific transcription factors expressed in the mother cell.