Naoyuki Fuse

Heterotrimeric G Proteins Regulate Daughter Cell Size Asymmetry in Drosophila Neuroblast Divisions

Cell division often generates unequally sized daughter cells by off-center cleavages, which are due to either displacement of mitotic spindles or their asymmetry. Drosophila neuroblasts predominantly use the latter mechanism to divide into a large apical neuroblast and a small basal ganglion mother cell (GMC), where the neural fate determinants segregate. Apically localized components regulate both the spindle asymmetry and the localization of the determinants. Here, we show that asymmetric spindle formation depends on signaling mediated by the G beta subunit of heterotrimeric G proteins. G beta 13F distributes throughout the neuroblast cortex. Its lack induces a large symmetric spindle and causes division into nearly equal-sized cells with normal segregation of the determinants. In contrast, elevated G beta 13F activity generates a small spindle, suggesting that this factor suppresses spindle development. Depletion of the apical components also results in the formation of a small symmetric spindle at metaphase. Therefore, the apical components and G beta 13F affect the mitotic spindle shape oppositely. We propose that differential activation of G beta signaling biases spindle development within neuroblasts and thereby causes asymmetric spindles. Furthermore, the multiple equal cleavages of G beta mutant neuroblasts accompany neural defects; this finding suggests indispensable roles of eccentric division in assuring the stem cell properties of neuroblasts.

Differential functions of G protein and Baz–aPKC signaling pathways in Drosophila neuroblast asymmetric division

Drosophila melanogaster neuroblasts (NBs) undergo asymmetric divisions during which cell-fate determinants localize asymmetrically, mitotic spindles orient along the apical–basal axis, and unequal-sized daughter cells appear. We identified here the first Drosophila mutant in the Gγ1 subunit of heterotrimeric G protein, which produces Gγ1 lacking its membrane anchor site and exhibits phenotypes identical to those of Gβ13F, including abnormal spindle asymmetry and spindle orientation in NB divisions. This mutant fails to bind Gβ13F to the membrane, indicating an essential role of cortical Gγ1–Gβ13F signaling in asymmetric divisions. In Gγ1 and Gβ13F mutant NBs, Pins–Gαi, which normally localize in the apical cortex, no longer distribute asymmetrically. However, the other apical components, Bazooka–atypical PKC–Par6–Inscuteable, still remain polarized and responsible for asymmetric Miranda localization, suggesting their dominant role in localizing cell-fate determinants. Further analysis of Gβγ and other mutants indicates a predominant role of Partner of Inscuteable–Gαi in spindle orientation. We thus suggest that the two apical signaling pathways have overlapping but different roles in asymmetric NB division.

Tre1 GPCR initiates germ cell transepithelial migration by regulating Drosophila melanogaster E-cadherin

Despite significant progress in identifying the guidance pathways that control cell migration, how a cell starts to move within an intact organism, acquires motility, and loses contact with its neighbors is poorly understood. We show that activation of the G protein–coupled receptor (GPCR) trapped in endoderm 1 (Tre1) directs the redistribution of the G protein Gβ as well as adherens junction proteins and Rho guanosine triphosphatase from the cell periphery to the lagging tail of germ cells at the onset of Drosophila melanogaster germ cell migration. Subsequently, Tre1 activity triggers germ cell dispersal and orients them toward the midgut for directed transepithelial migration. A transition toward invasive migration is also a prerequisite for metastasis formation, which often correlates with down-regulation of adhesion proteins. We show that uniform down-regulation of E-cadherin causes germ cell dispersal but is not sufficient for transepithelial migration in the absence of Tre1. Our findings therefore suggest a new mechanism for GPCR function that links cell polarity, modulation of cell adhesion, and invasion.

Genome features of "Dark-fly", a Drosophila line reared long-term in a dark environment.

Organisms are remarkably adapted to diverse environments by specialized metabolisms, morphology, or behaviors. To address the molecular mechanisms underlying environmental adaptation, we have utilized a Drosophila melanogaster line, termed “Dark-fly”, which has been maintained in constant dark conditions for 57 years (1400 generations). We found that Dark-fly exhibited higher fecundity in dark than in light conditions, indicating that Dark-fly possesses some traits advantageous in darkness. Using next-generation sequencing technology, we determined the whole genome sequence of Dark-fly and identified approximately 220,000 single nucleotide polymorphisms (SNPs) and 4,700 insertions or deletions (InDels) in the Dark-fly genome compared to the genome of the Oregon-R-S strain, a control strain. 1.8% of SNPs were classified as non-synonymous SNPs (nsSNPs: i.e., they alter the amino acid sequence of gene products). Among them, we detected 28 nonsense mutations (i.e., they produce a stop codon in the protein sequence) in the Dark-fly genome. These included genes encoding an olfactory receptor and a light receptor. We also searched runs of homozygosity (ROH) regions as putative regions selected during the population history, and found 21 ROH regions in the Dark-fly genome. We identified 241 genes carrying nsSNPs or InDels in the ROH regions. These include a cluster of alpha-esterase genes that are involved in detoxification processes. Furthermore, analysis of structural variants in the Dark-fly genome showed the deletion of a gene related to fatty acid metabolism. Our results revealed unique features of the Dark-fly genome and provided a list of potential candidate genes involved in environmental adaptation.

Heterotrimeric G protein signaling governs the cortical stability during apical constriction in Drosophila gastrulation

During gastrulation in Drosophila melanogaster, coordinated apical constriction of the cellular surface drives invagination of the mesoderm anlage. Forces generated by the cortical cytoskeletal network have a pivotal role in this cellular shape change. Here, we show that the organisation of cortical actin is essential for stabilisation of the cellular surface against contraction. We found that mutation of genes related to heterotrimeric G protein (HGP) signaling, such as Gβ13F, Gγ1, and ric-8, results in formation of blebs on the ventral cellular surface. The formation of blebs is caused by perturbation of cortical actin and induced by local surface contraction. HGP signaling mediated by two Gα subunits, Concertina and G-iα65A, constitutively regulates actin organisation. We propose that the organisation of cortical actin by HGP is required to reinforce the cortex so that the cells can endure hydrostatic stress during tissue folding.

Progenitor properties of symmetrically dividing Drosophila neuroblasts during embryonic and larval development.

Asymmetric cell division generates two daughter cells of differential gene expression and/or cell shape. Drosophila neuroblasts undergo typical asymmetric divisions with regard to both features; this is achieved by asymmetric segregation of cell fate determinants (such as Prospero) and also by asymmetric spindle formation. The loss of genes involved in these individual asymmetric processes has revealed the roles of each asymmetric feature in neurogenesis, yet little is known about the fate of the neuroblast progeny when asymmetric processes are blocked and the cells divide symmetrically. We genetically created such neuroblasts, and found that in embryos, they were initially mitotic and then gradually differentiated into neurons, frequently forming a clone of cells homogeneous in temporal identity. By contrast, larval neuroblasts with the same genotype continued to proliferate without differentiation. Our results indicate that asymmetric divisions govern lineage length and progeny fate, consequently generating neural diversity, while the progeny fate of symmetrically dividing neuroblasts depends on developmental stages, presumably reflecting differential activities of Prospero in the nucleus.

Gprk2 adjusts Fog signaling to organize cell movements in Drosophila gastrulation

Gastrulation of Drosophila melanogaster proceeds through sequential cell movements: ventral mesodermal (VM) cells are induced by secreted Fog protein to constrict their apical surfaces to form the ventral furrow, and subsequently lateral mesodermal (LM) cells involute toward the furrow. How these cell movements are organized remains elusive. Here, we observed that LM cells extended apical protrusions and then underwent accelerated involution movement, confirming that VM and LM cells display distinct cell morphologies and movements. In a mutant for the GPCR kinase Gprk2, apical constriction was expanded to all mesodermal cells and the involution movement was abolished. In addition, the mesodermal cells halted apical constriction prematurely in accordance with the aberrant accumulation of Myosin II. Epistasis analyses revealed that the Gprk2 mutant phenotypes were dependent on the fog gene. Overexpression of Gprk2 suppressed the effects of excess Cta, a downstream component of Fog signaling. Based on these findings, we propose that Gprk2 attenuates and tunes Fog-Cta signaling to prevent apical constriction in LM cells and to support appropriate apical constriction in VM cells. Thus, the two distinct cell movements in mesoderm invagination are not predetermined, but rather are organized by the adjustment of cell signaling.

Dynamics of Dark-Fly Genome Under Environmental Selections

Environmental adaptation is one of the most fundamental features of organisms. Modern genome science has identified some genes associated with adaptive traits of organisms, and has provided insights into environmental adaptation and evolution. However, how genes contribute to adaptive traits and how traits are selected under an environment in the course of evolution remain mostly unclear. To approach these issues, we utilize “Dark-fly”, a Drosophila melanogaster line maintained in constant dark conditions for more than 60 years. Our previous analysis identified 220,000 single nucleotide polymorphisms (SNPs) in the Dark-fly genome, but did not clarify which SNPs of Dark-fly are truly adaptive for living in the dark. We found here that Dark-fly dominated over the wild-type fly in a mixed population under dark conditions, and based on this domination we designed an experiment for genome reselection to identify adaptive genes of Dark-fly. For this experiment, large mixed populations of Dark-fly and the wild-type fly were maintained in light conditions or in dark conditions, and the frequencies of Dark-fly SNPs were compared between these populations across the whole genome. We thereby detected condition-dependent selections toward approximately 6% of the genome. In addition, we observed the time-course trajectory of SNP frequency in the mixed populations through generations 0, 22, and 49, which resulted in notable categorization of the selected SNPs into three types with different combinations of positive and negative selections. Our data provided a list of about 100 strong candidate genes associated with the adaptive traits of Dark-fly.

Verification of chicken Nanog as an epiblast marker and identification of chicken PouV as Pou5f3 by newly raised antibodies

Pluripotency is an important feature of early embryonic cells of multicellular organisms. Recent advances in stem cell research have shown that Nanog and Pou5f1 (Oct3/4) play important roles in mammalian pluripotency. However, whether these molecules exert conserved functions in other species remains unknown. Although the epiblast of the early chicken embryo would provide a useful experimental model, a lack of antibodies against chicken Nanog (cNanog) and chicken PouV/Pou5f3 (cPouV) proteins has hampered intensive investigation. Here we report newly raised polyclonal antibodies that specifically recognize cNanog and cPouV proteins. The specificity and sensitivity of the antibodies were validated by both western blotting and immunostaining with transfected 293T cells and chicken embryonic tissues. Immunohistochemistry using these antibodies revealed that cNanog protein was specifically localized in epiblastic cells and germ cells. In contrast, cPouV expression was seen almost ubiquitously. We also found that chicken epiblast‐derived colony‐forming cells that morphologically resemble mouse embryonic stem cells were cNanog‐positive, implying that these colony‐forming cells possess pluripotency. The anti‐cPouV antibody further enabled us to identify a previously unknown region at the N‐terminus of the cPouV protein containing a characteristic motif that is absent in mammalian Pou5f1. Thus, the antibodies raised in this study are useful tools for studying the functions of cNanog and cPouV at the protein level and the molecular mechanisms of chicken pluripotency.

Compound Eyes of Dark-fly

The ultrastructure of the compound eye of Dark- and control flies was examined. Flies of both groups reared with nutrient-poor Pearl’s medium exhibited a considerable degree of degeneration of the rhabdomeres. When they were reared with nutrient-rich standard cornmeal medium, the rhabdomeres of Dark-fly showed partial degeneration, while those of the control fly showed no degeneration. Thus, nutrients showed a far larger effect on the morphology of the eye than environmental light conditions, which slightly affected on eyes of flies that had experienced many generations of life without light.