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Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division

Nature Cell Biology volume 3, pages 50–57 (2001)Cite this article

Abstract

Cell-fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell divisions. In the Drosophila pupa, the pI sense organ precursor cell is polarized along the anterior–posterior axis of the fly and divides asymmetrically to generate a posterior pIIa cell and an anterior pIIb cell. The anterior pIIb cell specifically inherits the determinant Numb and the adaptor protein Partner of Numb (Pon). By labelling both the Pon crescent and the microtubules in living pupae, we show that determinants localize at the anterior cortex before mitotic-spindle formation, and that the spindle forms with random orientation and rotates to line up with the Pon crescent. By imaging living frizzled (fz) mutant pupae we show that Fz regulates the orientation of the polarity axis of pI, the initiation of spindle rotation and the unequal partitioning of determinants. We conclude that Fz participates in establishing the polarity of pI.

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Figure 1: Time-lapse imaging of the pI division.
Figure 2: Pon–GFP accumulates at the anterior cortex during prophase.
Figure 3: Centrosome movement and spindle dynamics in pI cells.
Figure 4: Spindle rotation directs the unequal segregation of Pon–GFP.
Figure 5: Centrosome movement and spindle dynamics in fz mutant pI cells.
Figure 6: Defective partitioning of Pon–GFP, Pon and Numb in fz mutant pupae.
Figure 7: A fz-numb interaction during the pIIa versus pIIb cell-fate decision.

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Acknowledgements

We thank P. Adler, W. Chia, Y.-N. Jan, T. Kanda, B.-W. Lu, N. Perrimon, J.-R. Martin, A. Martinez-Arias, E. Spana, G. M. Wahl and the Developmental Studies Hybridoma Bank for antibodies, plasmids and flies. We also thank G. Géraud and the confocal imaging facility of the Institut Jacques Monod, as well as the Electron Microscopy Facility of the University Paris 6 for use of confocal and SEM microsocopes. We thank A. Martinez-Arias, P. van Roessel and R. LeBorgne for critical reading. This work was supported in part by grants from the Centre National de la Recherche Scientifique (ATIPE), the Association pour la Recherche contre le Cancer (ARC 5951) and by the Wellcome Trust.

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Authors and Affiliations

  1. Ecole Normale Supérieure, UMR 8544, 46, rue d'Ulm, Paris, Cedex 05, 75230, France

    Yohanns Bellaïche, Michel Gho & François Schweisguth

  2. Wellcome/CRC Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge , CB2 1QR, UK

    Julia A. Kaltschmidt & Andrea H. Brand

Authors
  1. Yohanns Bellaïche
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  2. Michel Gho
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  3. Julia A. Kaltschmidt
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  4. Andrea H. Brand
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  5. François Schweisguth
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Corresponding author

Correspondence to François Schweisguth.

Supplementary information

Movie 1

pI divides unequally with a stereotyped planar orientation Wide-field time-lapse imaging of two dividing microchaete pI cells located in row 1. These pI cells, which specifically express gap-GFP under the control of neuP72, divide with a fixed orientation relative to the a-p axis, each generating a small anterior pIIb cell and a large posterior cell. Anterior is up. (MOV 50 kb)

Movie 2

Pon-GFP accumulates at the anterior cortex during prophase  Wide-field time-lapse imaging of a pI cell expressing both Histone2B-YFP and Pon-GFP. Progression into mitosis was followed using H2B-YFP to reveal chromatin condensation, alignment of the chromosomes onto the metaphase plate, chromosome migration to the pole and re-formation of daughter nuclei. Pon-GFP localises to the anterior cortex at late prophase. The perinuclear accumulation of Pon-GFP is masked by H2B-YFP. Anterior is up. (MOV 865 kb)

Movie 3

Centrosome movement and spindle dynamics in pI cells (1) Wide-field time-lapse imaging of pI cells expressing tau-GFP under the control of neuP72, showing centrosome separation and migration, mitotic spindle formation at prometaphase followed by a 45° rotation. In this movie, the centrosome that is eventually inherited by the posterior pIIb cell is closely associated with the nucleus and starts migrating around the nucleus before the other centrosome. The initial position of the centrosome relative to the nucleus does not, however, correlate with a specific spindle pole. Also, centrosome rotation starts prior to spindle formation. Anterior is up. (MOV 341 kb)

Movie 4

Centrosome movement and spindle dynamics in pI cells (2) Wide-field time-lapse imaging of pI cells expressing tau-GFP under the control of neuP72, showing a nearly 90° spindle rotation. Anterior is up. (MOV 311 kb)

Movie 5

Spindle rotation directs the unequal segregation of Pon-GFP (1)  Wide-field time-lapse imaging of a pI cell expressing both Pon-GFP and tau-GFP under the control of neuP72, showing formation of the Pon-GFP crescent prior to spindle formation and spindle rotation relative to the crescent of Pon-GFP. Anterior is up. (MOV 391 kb)

Movie 6

Spindle rotation directs the unequal segregation of Pon-GFP (2)  Wide-field time-lapse imaging of a pI cell expressing both Pon-GFP and tau-GFP under the control of neuP72, showing that the spindle rotates by 90¡ to line up with the anterior crescent of Pon-GFP. Anterior is up. (MOV 322 kb)

Movie 7

Spindle rotation and orientation in frizzled mutant pI cells (1)  Wide-field time-lapse imaging of a fzK21/fzKd4a mutant pI cell that expresses tau-GFP under the control of neuP72, showing bidirectional spindle rotation during metaphase. pI divides with an abnormal orientation relative to the a-p axis, and generates a small pIIb cell toward the posterior pole (bottom-right corner). Anterior is up. (MOV 149 kb)

Movie 8

Spindle rotation and orientation in frizzled mutant pI cells (2)  Confocal time-lapse imaging of a fzK21/fzKd4a mutant pI cell that expresses tau-GFP under the control of neuP72. Anterior is up. (MOV 29 kb)

Movie 9

Pon-GFP accumulates at the anterior cortex during prophase  Confocal time-lapse imaging of a control neuP72 fzKd4a UAS-Pon-GFP / + pI cell expressing Pon-GFP under the control of neuP72. Pon-GFP is intially found around the nucleus and in basal cytoplasmic extensions that retract during prophase. Pon-GFP accumulates at the anterior pole during prophase, and segregates into the anterior pIIb cell. Anterior is up. (MOV 32 kb)

Movie 10

Unequal segregation of Pon-GFP in frizzled mutant pI cells (1)  Confocal time-lapse imaging of a fzK21 / neuP72 fzKd4a UAS-Pon-GFP mutant pI cells. Pon-GFP forms a randomly positioned crescent in fz mutant pI cells. In this movie, Pon-GFP localises in an abnormally large lateral crescent, and cytokinesis bisects this Pon-GFP crescent, thereby leading to the segregation of Pon-GFP to both daughter cells. Anterior is up. (MOV 566 kb)

Movie 11

Unequal segregation of Pon-GFP in frizzled mutant pI cells (2)  Wide-field time-lapse imaging of a fzK21 / neuP72 fzKd4a UAS-Pon-GFP mutant pI cells. Pon-GFP forms a randomly positioned crescent in fz mutant pI cells. In this movie, Pon-GFP localises in an abnormally large posterior crescent and cytokinesis bisects this Pon-GFP crescent, thereby leading to the segregation of Pon-GFP to both daughter cells. Anterior is up. (MOV 1106 kb)

Movie 12

Unequal segregation of Pon-GFP in frizzled mutant pI cells (3)  Wide-field time-lapse imaging of a fzK21 UAS-tau-GFP / neuP72 fzKd4a UAS-Pon-GFP mutant pI cells. Pon-GFP localises asymmetrically prior to spindle formation and forms a large posterior crescent. In this pI cell, the spindle does not rotate significantly, and does not line up with the center of the Pon-GFP crescent, thereby leading to the partial mis-partitionning of Pon-GFP to both daughter cells. Anterior is up. (MOV 230 kb)

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Bellaïche, Y., Gho, M., Kaltschmidt, J. et al. Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division. Nat Cell Biol 3, 50–57 (2001). https://doi.org/10.1038/35050558

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