Aster repulsion drives short-ranged ordering in the Drosophila syncytial blastoderm

Biological systems are highly complex, yet notably ordered structures can emerge. During syncytial stage development of the Drosophila melanogaster embryo, nuclei synchronously divide for nine cycles within a single cell, after which most of the nuclei reach the cell cortex. The arrival of nuclei to the cortex occurs with remarkable positional order, which is important for subsequent cellularisation and morphological transformations. Yet, the mechanical principles underlying this lattice-like positional order of nuclei remain untested. Here, utilising quantification of nuclei position and division orientation together with embryo explants we show that short-ranged repulsive interactions between microtubule asters ensure the regular distribution and maintenance of nuclear positions in the embryo. Such ordered nuclear positioning still occurs with the loss of actin caps and even the loss of the nuclei themselves; the asters can self-organise with similar distribution to nuclei in the wild-type embryo. The explant assay enabled us to deduce the nature of the mechanical interaction between pairs of nuclei. We used this to predict how the nuclear division axis orientation changes upon nucleus removal from the embryo cortex, which we confirmed in vivo with laser ablation. Overall, we show that short-ranged microtubule-mediated repulsive interactions between asters are important for ordering in the early Drosophila embryo and minimising positional irregularity.

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Aster repulsion drives local ordering in an active system

10.1101/2020.06.04.133579 ◽

2020 ◽

Author(s):

Jorge de-Carvalho ◽

Sham Tlili ◽

Lars Hufnagel ◽

Timothy E. Saunders ◽

Ivo A. Telley

Keyword(s):

Nuclear Division ◽

Ex Vivo ◽

Cell Cortex ◽

Active System ◽

Axis Orientation ◽

Local Ordering ◽

Material State ◽

Division Axis ◽

Morphological Transformations ◽

Open Question

AbstractBiological systems are a form of active matter, which often undergo rapid changes in their material state, e.g. liquid to solid transitions. Yet, such systems often also display remarkably ordered structures. It remains an open question as to how local ordering occurs within active systems. Here, we utilise the rapid early development of Drosophila melanogaster embryos to uncover the mechanisms driving short-ranged order. During syncytial stage, nuclei synchronously divide (within a single cell defined by the ellipsoidal eggshell) for nine cycles after which most of the nuclei reach the cell cortex. Despite the rapid nuclear division and repositioning, the spatial pattern of nuclei at the cortex is highly regular. Such precision is important for subsequent cellularisation and morphological transformations. We utilise ex vivo explants and mutant embryos to reveal that microtubule asters ensure the regular distribution and maintenance of nuclear positions in the embryo. For large networks of nuclei, such as in the embryo, we predict – and experimentally verify – the formation of force chains. The ex vivo extracts enabled us to deduce the force potential between single asters. We use this to predict how the nuclear division axis orientation in small ex vivo systems depend on aster number. Finally, we demonstrate that, upon nucleus removal from the cortex, microtubule force potentials can reorient subsequent nuclear divisions to minimise the size of pattern defects. Overall, we show that short-ranged microtubule-mediated repulsive interactions between asters can drive ordering within an active system.

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The Rap1–Rgl–Ral signaling network regulates neuroblast cortical polarity and spindle orientation

The Journal of Cell Biology ◽

10.1083/jcb.201108112 ◽

2011 ◽

Vol 195 (4) ◽

pp. 553-562 ◽

Cited By ~ 31

Author(s):

Ana Carmena ◽

Aljona Makarova ◽

Stephan Speicher

Keyword(s):

Cell Fate ◽

Asymmetric Cell Division ◽

Signaling Network ◽

Spindle Orientation ◽

Nucleotide Exchange ◽

Loss Of Function ◽

Axis Orientation ◽

Division Axis ◽

Guanosine Triphosphatase

A crucial first step in asymmetric cell division is to establish an axis of cell polarity along which the mitotic spindle aligns. Drosophila melanogaster neural stem cells, called neuroblasts (NBs), divide asymmetrically through intrinsic polarity cues, which regulate spindle orientation and cortical polarity. In this paper, we show that the Ras-like small guanosine triphosphatase Rap1 signals through the Ral guanine nucleotide exchange factor Rgl and the PDZ protein Canoe (Cno; AF-6/Afadin in vertebrates) to modulate the NB division axis and its apicobasal cortical polarity. Rap1 is slightly enriched at the apical pole of metaphase/anaphase NBs and was found in a complex with atypical protein kinase C and Par6 in vivo. Loss of function and gain of function of Rap1, Rgl, and Ral proteins disrupt the mitotic axis orientation, the localization of Cno and Mushroom body defect, and the localization of cell fate determinants. We propose that the Rap1–Rgl–Ral signaling network is a novel mechanism that cooperates with other intrinsic polarity cues to modulate asymmetric NB division.

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Quantitative Analysis of Gene Function in the Drosophila Embryo

Genetics ◽

10.1093/genetics/154.1.273 ◽

2000 ◽

Vol 154 (1) ◽

pp. 273-284

Author(s):

William D Tracey ◽

Xiangqun Ning ◽

Martin Klingler ◽

Sunita G Kramer ◽

J Peter Gergen

Keyword(s):

Gene Function ◽

Model Systems ◽

Drosophila Embryo ◽

Direct Role ◽

Developmental Context ◽

Maternal Mrna ◽

Early Drosophila Embryo ◽

Segment Polarity ◽

Pair Rule

Abstract The specific functions of gene products frequently depend on the developmental context in which they are expressed. Thus, studies on gene function will benefit from systems that allow for manipulation of gene expression within model systems where the developmental context is well defined. Here we describe a system that allows for genetically controlled overexpression of any gene of interest under normal physiological conditions in the early Drosophila embryo. This regulated expression is achieved through the use of Drosophila lines that express a maternal mRNA for the yeast transcription factor GAL4. Embryos derived from females that express GAL4 maternally activate GAL4-dependent UAS transgenes at uniform levels throughout the embryo during the blastoderm stage of embryogenesis. The expression levels can be quantitatively manipulated through the use of lines that have different levels of maternal GAL4 activity. Specific phenotypes are produced by expression of a number of different developmental regulators with this system, including genes that normally do not function during Drosophila embryogenesis. Analysis of the response to overexpression of runt provides evidence that this pair-rule segmentation gene has a direct role in repressing transcription of the segment-polarity gene engrailed. The maternal GAL4 system will have applications both for the measurement of gene activity in reverse genetic experiments as well as for the identification of genetic factors that have quantitative effects on gene function in vivo.

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Regulated nuclear import of the Drosophila rel protein dorsal: structure-function analysis.

Molecular and Cellular Biology ◽

10.1128/mcb.16.3.1103 ◽

1996 ◽

Vol 16 (3) ◽

pp. 1103-1114 ◽

Cited By ~ 22

Author(s):

S Govind ◽

E Drier ◽

L H Huang ◽

R Steward

Keyword(s):

Amino Acids ◽

Structure Function ◽

Nuclear Localization ◽

Nuclear Import ◽

Function Analysis ◽

Drosophila Embryo ◽

Nuclear Targeting ◽

Homology Region ◽

Early Drosophila Embryo

The formation of a gradient of nuclear Dorsal protein in the early Drosophila embryo is the last step in a maternally encoded dorsal-ventral signal transduction pathway. This gradient is formed in response to a ventral signal, which leads to the dissociation of cytoplasmic Dorsal from the I kappa B homolog Cactus. Free Dorsal is then targeted to the nucleus. Dorsal is a Rel-family transcription factor. Signal-dependent nuclear localization characterizes the regulation of Rel proteins. In order to identify regions of Dorsal that are essential for its homodimerization, nuclear targeting, and interaction with Cactus, we have performed an in vivo structure-function analysis. Our results show that all these functions are carried out by regions within the conserved Rel-homology region of Dorsal. The C-terminal divergent half of Dorsal is dispensable for its selective nuclear import. A basic stretch of 6 amino acids at the C terminus of the Rel-homology region is necessary for nuclear localization. This nuclear localization signal is not required for Cactus binding. Removal of the N-terminal 40 amino acids abolished the nuclear import of Dorsal, uncovering a potentially novel function for this highly conserved region.

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Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo.

The Journal of Cell Biology ◽

10.1083/jcb.109.6.2977 ◽

1989 ◽

Vol 109 (6) ◽

pp. 2977-2991 ◽

Cited By ~ 80

Author(s):

D R Kellogg ◽

C M Field ◽

B M Alberts

Keyword(s):

Affinity Chromatography ◽

Mitotic Spindle ◽

Polyclonal Antibodies ◽

Drosophila Embryo ◽

Microtubule Cytoskeleton ◽

Microtubule Associated Proteins ◽

Early Drosophila Embryo ◽

Individual Mouse ◽

Associated Proteins

We have developed affinity chromatography methods for the isolation of microtubule-associated proteins (MAPs) from soluble cytoplasmic extracts and have used them to analyze the cytoskeleton of the early Drosophila embryo. More than 50 Drosophila embryo proteins bind to microtubule affinity columns. To begin to characterize these proteins, we have generated individual mouse polyclonal antibodies that specifically recognize 24 of them. As judged by immunofluorescence, some of the antigens localize to the mitotic spindle in the early Drosophila embryo, while others are present in centrosomes, kinetochores, subsets of microtubules, or a combination of these structures. Since 20 of the 24 antibodies stain microtubule structures, it is likely that most of the proteins that bind to our columns are associated with microtubules in vivo. Very few MAPS seem to be identically localized in the cell, indicating that the microtubule cytoskeleton is remarkably complex.

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Morphogenetic forces planar polarize LGN/Pins in the embryonic head during Drosophila gastrulation

10.1101/2022.01.07.475359 ◽

2022 ◽

Author(s):

Jaclyn M Camuglia ◽

Soline Chanet ◽

Adam C Martin

Keyword(s):

Planar Cell Polarity ◽

Adherens Junction ◽

Drosophila Embryo ◽

Spindle Orientation ◽

Planar Polarity ◽

Ventral Furrow ◽

Adherens Junction Protein ◽

Early Drosophila Embryo ◽

Junction Protein

Spindle orientation is often achieved by a complex of Pins/LGN, Mud/NuMa, Gαi, and Dynein, which interacts with astral microtubules to rotate the spindle. Cortical Pins/LGN recruitment serves as a critical step in this process. Here, we identify Pins-mediated planar cell polarized divisions in several of the mitotic domains of the early Drosophila embryo. We found that neither planar cell polarity pathways nor planar polarized myosin localization determined division orientation; instead, our findings strongly suggest that Pins planar polarity and force generated from mesoderm invagination are important. Disrupting Pins polarity via overexpression of a myristoylated version of Pins caused randomized division angles. We found that disrupting forces through chemical inhibitors, laser ablation, and depletion of an adherens junction protein disrupted Pins planar polarity and spindle orientation. Furthermore, snail depletion, which abrogates ventral furrow forces, disrupted Pins polarization and spindle orientation, suggesting that morphogenetic movements and resulting forces transmitted through the tissue can polarize Pins and orient division. Thus, morphogenetic forces associated with mesoderm invagination result in planar polarized Pins to mediate division orientation at a distant region of the embryo during morphogenesis. To our knowledge, this is the first in vivo example where mechanical force has been shown to polarize Pins to mediate division orientation.

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Cyclin A and B functions in the early Drosophila embryo

Development ◽

10.1242/dev.126.23.5505 ◽

1999 ◽

Vol 126 (23) ◽

pp. 5505-5513 ◽

Cited By ~ 5

Author(s):

L.A. Stiffler ◽

J.Y. Ji ◽

S. Trautmann ◽

C. Trusty ◽

G. Schubiger

Keyword(s):

Cyclin A ◽

Cyclin B ◽

Drosophila Embryo ◽

Late Prophase ◽

Early Drosophila Embryo ◽

Nuclear Events ◽

The Relationship ◽

Maternal Gene ◽

Drosophila Embryos

In eukaryotes, mitotic cyclins localize differently in the cell and regulate different aspects of the cell cycle. We investigated the relationship between subcellular localization of cyclins A and B and their functions in syncytial preblastoderm Drosophila embryos. During early embryonic cycles, cyclin A was always concentrated in the nucleus and present at a low level in the cytoplasm. Cyclin B was predominantly cytoplasmic, and localized within nuclei only during late prophase. Also, cyclin B colocalized with metaphase but not anaphase spindle microtubules. We changed maternal gene doses of cyclins A and B to test their functions in preblastoderm embryos. We observed that increasing doses of cyclin B increased cyclin B-Cdk1 activity, which correlated with shorter microtubules and slower microtubule-dependent nuclear movements. This provides in vivo evidence that cyclin B-Cdk1 regulates microtubule dynamics. In addition, the overall duration of the early nuclear cycles was affected by cyclin A but not cyclin B levels. Taken together, our observations support the hypothesis that cyclin B regulates cytoskeletal changes while cyclin A regulates the nuclear cycles. Varying the relative levels of cyclins A and B uncoupled the cytoskeletal and nuclear events, so we speculate that a balance of cyclins is necessary for proper coordination during these embryonic cycles.

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Geometric constraints alter cell arrangements within curved epithelial tissues

Molecular Biology of the Cell ◽

10.1091/mbc.e17-01-0060 ◽

2017 ◽

Vol 28 (25) ◽

pp. 3582-3594 ◽

Cited By ~ 13

Author(s):

Jean-Francois Rupprecht ◽

Kok Haur Ong ◽

Jianmin Yin ◽

Anqi Huang ◽

Huy-Hong-Quan Dinh ◽

...

Keyword(s):

Cell Morphology ◽

Three Dimensional ◽

Quantitative Information ◽

Geometric Constraints ◽

Three Dimensions ◽

Drosophila Embryo ◽

Anterior Pole ◽

Epithelial Tissues ◽

Early Drosophila Embryo

Organ and tissue formation are complex three-dimensional processes involving cell division, growth, migration, and rearrangement, all of which occur within physically constrained regions. However, analyzing such processes in three dimensions in vivo is challenging. Here, we focus on the process of cellularization in the anterior pole of the early Drosophila embryo to explore how cells compete for space under geometric constraints. Using microfluidics combined with fluorescence microscopy, we extract quantitative information on the three-dimensional epithelial cell morphology. We observed a cellular membrane rearrangement in which cells exchange neighbors along the apical-basal axis. Such apical-to-basal neighbor exchanges were observed more frequently in the anterior pole than in the embryo trunk. Furthermore, cells within the anterior pole skewed toward the trunk along their long axis relative to the embryo surface, with maximum skew on the ventral side. We constructed a vertex model for cells in a curved environment. We could reproduce the observed cellular skew in both wild-type embryos and embryos with distorted morphology. Further, such modeling showed that cell rearrangements were more likely in ellipsoidal, compared with cylindrical, geometry. Overall, we demonstrate that geometric constraints can influence three-dimensional cell morphology and packing within epithelial tissues.

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