Lgl, Pins and aPKC regulate neuroblast self-renewal versus differentiation

Abstract

How a cell chooses to proliferate or to differentiate is an important issue in stem cell and cancer biology. Drosophila neuroblasts undergo self-renewal with every cell division, producing another neuroblast and a differentiating daughter cell, but the mechanisms controlling the self-renewal/differentiation decision are poorly understood. Here we tested whether cell polarity genes, known to regulate embryonic neuroblast asymmetric cell division1, also regulate neuroblast self-renewal. Clonal analysis in larval brains showed that pins mutant neuroblasts rapidly fail to self-renew, whereas lethal giant larvae (lgl) mutant neuroblasts generate multiple neuroblasts. Notably, lgl pins double mutant neuroblasts all divide symmetrically to self-renew, filling the brain with neuroblasts at the expense of neurons. The lgl pins neuroblasts show ectopic cortical localization of atypical protein kinase C (aPKC), and a decrease in aPKC expression reduces neuroblast numbers, suggesting that aPKC promotes neuroblast self-renewal. In support of this hypothesis, neuroblast-specific overexpression of membrane-targeted aPKC, but not a kinase-dead version, induces ectopic neuroblast self-renewal. We conclude that cortical aPKC kinase activity is a potent inducer of neuroblast self-renewal.

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Figure 1: lgl and pins regulate larval neuroblast self-renewal.
Figure 2: lgl pins double mutants have ectopic neuroblasts and fewer neurons.
Figure 3: Lgl and Pins regulate aPKC localization in larval neuroblasts.
Figure 4: Overexpression of cortical aPKC promotes neuroblast self-renewal.

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Acknowledgements

We thank J. Knoblich, S. Campuzano, B. Chia, J. Skeath and B. Holmgren for fly stocks and/or antibody reagents; B. Bowerman, J. Eisen, K. Siller and S. Siegrist for comments on the manuscript; and C. Chabu for discussion. C.-Y.L. is supported by a Damon Runynon postdoctoral fellowship. C.Q.D. is supported by the Howard Hughes Medical Institute, where he is an Investigator.

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Correspondence to Chris Q. Doe.

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Supplementary information

Supplementary Figure 1

lgl and pins regulate larval neuroblast numbers. (PDF 132 kb)

Supplementary Figure 2

Apoptosis is not increased in pins mutant brains. (PDF 59 kb)

Supplementary Figure 3

A comparison of neuroblast and GMC markers reveals that lgl pins mutant brains have supernumerary neuroblasts. (PDF 326 kb)

Supplementary Figure 4

aPKC is required for neuroblast self-renewal in lgl mutants. (PDF 120 kb)

Supplementary Data

Gene/protein list (DOC 19 kb)

Supplementary Methods

Additional description of the methods used in this study. (DOC 23 kb)

Supplementary Movie1

Thousands of differentiating GMCs and neurons rapidly down-regulate neuroblast markers and express nuclear Prospero and/or Elav. (MOV 2660 kb)

Supplementary Movie 2

There was a clear increase in neuroblast number in lgl and dlg mutants; and also supernumerary neuroblasts at all stages examined. All extra neuroblasts expressed Deadpan and Miranda neuroblast markers and were proliferative based on their ability to incorporate BrdU. (MOV 2750 kb)

Supplementary Movie3

Gαi zygotic mutants had a complex phenotype, but pins zygotic mutants showed a striking decrease in neuroblast number. (MOV 2450 kb)

Supplementary Movie 4

A novel phenotype was detected, in which the larval brain was full of cells expressing the neuroblast markers Worniu, Miranda and Deadpan, and lacking expression of the neuronal marker Elav. (MOV 2209 kb)

Supplementary Movie 5

Neuroblast-specific expression of aPKC targeted to the plasma membrane with a CAAX prenylation motif (UAS-aPKCCAAXWT) resulted in ectopic cortical aPKC localization, loss of cortical Miranda and a massive increase in the number of neuroblasts. (MOV 2686 kb)

Supplementary Movie 6

Neuroblast-specific expression of aPKC targeted to the plasma membrane with a CAAX prenylation motif (UAS-aPKCCAAXWT) resulted in ectopic cortical aPKC localization, loss of cortical Miranda and a massive increase in the number of neuroblasts. These effects were not observed following overexpression of wild type aPKC or a membrane-targeted kinase-dead aPKC (UAS-aPKCCAAXKD). (MOV 2431 kb)

Supplementary Figure Legends

Text to accompany the above Supplementary Figures. (DOC 21 kb)

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Lee, C., Robinson, K. & Doe, C. Lgl, Pins and aPKC regulate neuroblast self-renewal versus differentiation. Nature 439, 594–598 (2006). https://doi.org/10.1038/nature04299

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