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The microcephaly protein Asp regulates neuroepithelium morphogenesis by controlling the spatial distribution of myosin II

Nature Cell Biology volume 15, pages 1294–1306 (2013)Cite this article

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Abstract

Mutations in ASPM are the most frequent cause of microcephaly, a disorder characterized by reduced brain size at birth. ASPM is recognized as a major regulator of brain size, yet its role during neural development remains poorly understood. Moreover, the role of ASPM proteins in invertebrate brain morphogenesis has never been investigated. Here, we characterized the function of the Drosophila ASPM orthologue, Asp, and found that asp mutants present severe defects in brain size and neuroepithelium morphogenesis. We show that size reduction depends on the mitotic function of Asp, whereas regulation of tissue shape depends on an uncharacterized function. Asp interacts with myosin II regulating its polarized distribution along the apico-basal axis. In the absence of Asp, mislocalization of myosin II results in interkinetic nuclear migration and tissue architecture defects. We propose that Asp regulates neuroepithelium morphogenesis through myosin-II-mediated structural and mechanical processes to maintain force balance and tissue cohesiveness.

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Figure 1: Neuroepithelium morphogenesis is impaired in asp mutant brains.
Figure 2: asp mutant brains exhibit high levels of apoptosis.
Figure 3: asp mutants exhibit defects in spindle position.
Figure 4: asp mutants exhibit defects in INM.
Figure 5: Myo-II is mislocalized in asp mutants.
Figure 6: Asp regulates apico-basal distribution of tension and epithelial cohesiveness by promoting polarized localization of myo-II.
Figure 7: Asp localizes at the basal membrane together with myo-II and interacts with MHC in vivo.
Figure 8: A model for Asp functions in neuroepithelium morphogenesis in Drosophila.

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Acknowledgements

We thank Y. Bellaiche (Institut Curie, France), A. Brand (Gurdon Institute, UK), A. Carmena (UMH, Spain), C. Doe (University of Oregon, USA), B. Egger (University of Fribourg, Switzerland), M. Gatti (University of Roma, Italy), C. Gonzalez (IRB, Spain), R. Karess (IJM, France), E. Knust (MPI, Germany), J. C. Lehner (IMLS, Switzerland), S. Heidmann (University of Bayreuth, Germany), F. Matzuzaki (Riken, Japan), J. Skeath (Washington University, USA), A. Wodarz (Göttingen University, Germany), Y. Yan (UCSF, USA), the Bloomington Stock Center, DSHB and the Antibody platform Institut Curie for stocks and reagents; V. Fraisier, L. Sengmanivong, F. Waharte, O. Leroy, C. Gueudry of the Imaging facility (PICT-IBISA) and the Nikon Center at the I. Curie for valuable help and advice on image acquisition and processing; A. M. Wehenkel, Z. Wang and A. Houdusse for advice on biochemistry; and F. Bosveld, Y. Bellaiche, D. Delacour, A. Echard, B. Egger, M. Piel, N. Minc, K. Roper, C. Norden, E. Gomes, D. Gogendeau, V. Marthiens, D. Sabino, D. Gambarotto, M. Nano, A. Booth and C. Janke for helpful discussions and critical comments on the manuscript. We thank the Institut Curie, EMBO (ALTF 923-2008), FRM (SPF20101220951) and the ERC starting grant for post-doctoral support (M.A.R.), and the MRC UK for postdoctoral support (L.S-P.). This work was supported by an ERC grant CentroStemCancer 242598, an FRM installation grant, an ATIP grant, the Institut Curie and the CNRS. The Basto laboratory is a member of the Labex CelTisPhyBio.

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  1. Gaelle le Dez

    Present address: Present address: IGDR, UMR 6290 -Universite de Rennes, F-35043 Rennes, France,

Authors and Affiliations

  1. UMR144, CNRS- Institut Curie, 12 rue Lhomond, 75005 Paris, France

    Maria A. Rujano, Carole Pennetier, Gaelle le Dez & Renata Basto

  2. MRC Functional Genomics Unit, University of Oxford, Oxford OX1 3QX, UK

    Luis Sanchez-Pulido

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Contributions

M.A.R. and R.B. conceived the project, analysed the data and wrote the manuscript. M.A.R. did most of the experimental procedures. L.S-P. performed the bioinformatics sequence analysis. C.P. and G.l.D. generated tools. R.B. supervised the project.

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Correspondence to Renata Basto.

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Integrated supplementary information

Supplementary Figure 1 Analysis of neurogenesis in asp mutant brain.

(A) WT (top) and asp (bottom) third instar larvae brain lobes stained for Dachshund to visualize the lamina neurons in the optic lobe. Scale bar: 30 μm. (B) WT (top) and asp (bottom) third instar larvae brains stained for Synapsin to visualize the synapses in the brain. Scale bar: 100 μm. (C) WT (top) and asp (bottom) third instar larvae brain lobes stained for Elav, a pan neuronal marker, to visualize the entire neuronal population. Scale bar: 30 μm.

Supplementary Figure 2 Neuroepithelium morphogenesis at early L3 stages.

(A) WT (top) and asp mutant (bottom) mid third instar larval brains stained with Armadillo (arm) (left panel and in blue in the merged panel) to label the neuroepithelium, Deadpan (Dpn) (red in the merged panel), which labels NBs, and L’Sc (green in the merged panel) labels the TZ. Top view representing the anterior surface view (left) and a cross-section (right) are shown. Scale bar: 30 μm. (B–C) aspE3/Def (B) and aspE3/asp1 (C) trans-heterozygous mid third instar larval brains stained with arm (blue in the merged panel) and Dpn (red in the merged panel). Scale bar: 0 μm.

Supplementary Figure 3 Establishment of epithelial identity is not affected in asp mutants.

(A) Schematic representation of epithelial polarity domains in Drosophila. (B) WT (left) and asp (right) neuroepithelial cells stained for the adherens junction marker Armadillo. (C) WT (top) and asp (bottom) neuroepithelial cells stained for Dlg, Par3 and Par6 (shown in blue, red and green respectively in the merged panel). (D) WT (top) and asp (bottom) neuroepithelial cells stained for Actin, PatJ and DE-Cad (shown in blue, green and red respectively in the merged panel). (E) WT (top) asp (bottom) neuroepithelial cells stained for βPS-Integrin and DE-Cad (shown in green and red respectively in the merged panel). Scale bars: 5 μm.

Supplementary Figure 4 Abnormal chromosome segregation occurs in asp mutant neuroepithelial cells.

(A, B) Still images of time lapse movies of WT (A) and asp mutant (B) dividing neuroepithelial cells expressing α-Tubulin-GFP (green in merged panel) and Histone-RFP (red in merged panel and shown separately in bottom panels) to label spindle MTs and DNA respectively. Apical is up and illustrated with a dashed red line in the Hist-RFP separate panels. Arrowheads indicate the dividing nuclei. In the asp mutant (B) daughter cells with different DNA content is noticed. Scale bars: 5 μm.

Supplementary Figure 5 Analysis of cell death by apoptosis in asp.

(A) asp neuroepithelium stained for Caspase-3 to label apoptotic cells (red in merged panels), Deadpan to label neuroblasts (green in merged panels) and Armadillo to label the neuroepithelium (blue in merged panels). The white dash line labels the neuroepithelium. Scale bars: 10 μm. (B) asp neuroepithelium stained for Caspase-3 to label apoptotic cells (red in merged panels), L’sc to label cells in the transition zone (green in merged panels) and Armadillo to label the neuroepithelium (blue in merged panels). Scale bars: 10 μm. (C) asp optic lobe stained for Caspase-3 to label apoptotic cells (red in merged panels), and ELAV to label neurons (green in merged panels). Scale bars: 30 μm.

Supplementary Figure 6 Oblique/perpendicular spindle positioning in Drosophila neuroepithelium.

(A) Low magnification still images of a time lapse movie of WT L3 neuroepithelium expressing α-Tubulin-RFP (red in merged panel) and Sqh-GFP (green in merged panel and shown separately in bottom panels) to label spindle MTs and myo-II respectively. Arrows indicate the dividing cell in the middle of the neuroepithelium with a 44° angle relative to the plane of the epithelium. At t = 00:00 the cell is in metaphase and at t = 06:00 the cell in anaphase. These images are consecutive to the image presented in Fig. 5a which will correspond to t = −06:00. Note that the spindle has rotated from a perfect parallel from t = −06:00 to t = 00:00. Scale bar: 10 μm. (B) High magnification of still images of a time lapse movie of WT L3 neuroepithelium expressing α-Tubulin-RFP (red in merged panel) and Sqh-GFP (green in merged panel and shown separately in bottom panels) to label spindle MTs and myo-II respectively. The mitotic spindle is positioned at a 41° angle relative to the plane of the epithelium in the dividing cell. At t = 00:00 the cell is in late-metaphase and at t = 04:30 the cell in anaphase. Scale bar: 10 μm. (C) WT neuroepithelium stained for Cnn to label mitotic centrosomes (green in merged panel and shown separately in bottom left panel), Deadpan to label neuroblasts (red in merged panels and shown separately in bottom right panel) and Armadillo to label the neuroepithelium (shown separately in upper right panel). The DNA is in blue in the merged panel. Scale bars: 10 μm.

Supplementary Figure 7 Analysis of myo-II phosphorylation mutants in neuroepithelium morphogenesis.

(A-B) SqhEE, asp (A) and SqhAA, asp (B) mutant brains stained with Armadillo to visualize the NE and Hoescht (red in merged panels) to label DNA. Low magnification images showing the anterior surface (top view, left), and a cross-section (middle) view of the third instar larval brains. A higher magnification of the NE is shown in the NE overview panel (right). Scale bar: 30 μm in top and cross section views and 10 μm in overview. (C) Quantification of the NE length in the apical-basal axis (bars) and the length of the basal process of dividing nuclei (dot plots) in SqhEE (n = 28 and n = 25 cells for cell length and basal process length respectively), SqhEE, asp (n = 66 and n = 38 for cell length and basal process length respectively), SqhAA (n = 57 and n = 20 cells for cell length and basal process length respectively) and SqhAA, asp (n = 100 and n = 48 cells for cell length and basal process length respectively). This plot should be compared to plot shown in Fig. 4d for WT and asp. Statistical significance was assessed by a two-tailed unpaired t-Test.

Supplementary Figure 8 Protein sequence analysis of the ASP family.

(A) Domain architecture of Drosophila Asp (top), mouse ASPM (middle) and human ASPM (bottom) proteins. We identified three consecutive calponin homology domains (blue) in human ASPM (the first two are conserved in Drosophila ASP), with highly significant HHpred E-values (see Methods): 7.4E-3, 7.2E-19 and 1.9E-20 for calponin homology domains one, two and three, respectively. In agreement with previous ASP protein sequence analysis, we also identified in the ASP family: (I) an N-terminal Hydin domain (green)1, (II) IQ repeats (red) identified by SMART domain database2 and, (III) a C-terminal region highly likely to contain Heat/Armadillo-like repeats (violet)3,4. Below, representative multiple sequence alignments of evolutionary conserved regions in ASP family. Alignments were produced using a combination of T-Coffee5 and the profile-to-profile alignment program HHalign6. The numbering after the protein name indicates the domain-repeat number when more than one is detected in the same sequence (in calponin homology domain and IQ repeats alignments). The limits of the protein sequences included in the alignment are indicated by the residue positions provided at each side. The alignment was presented with the program Belvu7 using a coloring scheme indicating the average BLOSUM62 scores (which are correlated with amino acid conservation) of each alignment column: red (>2.5), violet (between 2.5 and 1) and light yellow (between 1 and 0.2). Sequences are named according to their UniProt identifications8. Species abbreviations: AEDAE, Aedes aegypti; ANOGA, Anopheles gambiae; DANRE, Danio rerio; DROGR, Drosophila grimshawi; DROME, Drosophila melanogaster; HUMAN, Homo sapiens; MOUSE, Mus musculus; XENTR, Xenopus tropicalis. (B) Dendrogram and domain architecture for calponin homology (CH) domains-containing proteins ASPM and ASP. The dendrogram was calculated using the CH domain alignment shown in (A) with the neighbor-joining method9 and edited with Treetool. The scale bar shows the average number of amino acid substitutions per site (0.05). For the sake of simplicity only CH domains from human ASPM, mouse ASPM and D. melanogaster ASP are shown. The dashed boxes indicate the protein regions maintained in the mutant allele aspE3 of Drosophila Asp and the truncated ASPM (Aspm 1-7) from Pulvers et al., 2010. Note that Aspm 1-7 conserves an intact CH1. (C) CH1 structural model from D. melanogaster ASP was created using Modeller10 based on the human alpha-actinin CH domain structure (PDB: 1WKU; ref. 11). The model is presented using Pymol (http://www.pymol.org) and shows that the aspE3 (1-721) product does not include a functional CH1 domain (612-748), since the C-terminal alpha-helix (in red) is essential for the folding of the CH domain.

Supplementary Figure 9 Full gel scan.

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Mitosis and chromosome segregation in WT neuroepithelial cells.

Movie of WT dividing neuroepithelial cell expressing α -Tubulin-GFP (green in merged panel and shown separately in right panel) and Histone-RFP (red in merged panel and shown separately in middle panel) to label spindle MTs and DNA respectively. Time is given in minutes. (AVI 558 kb)

Mitosis and chromosome segregation in asp mutant neuroepithelial cells.

Movie of asp dividing neuroepithelial cell expressing α-Tubulin-GFP (green in merged panel and shown separately in right panel) and Histone-RFP (red in merged panel and shown separately in middle panel) to label spindle MTs and DNA respectively. Time is given in minutes. (AVI 1050 kb)

Parallel spindle positioning in WT neuroepithelial cells.

Movies of three WT dividing neuroepithelial cells expressing α-Tubulin-RFP (red in merged panel and shown separately in middle panels) to label spindle MTs and Pon-GFP (green in merged panel and shown separately in bottom panel) used as a polarity marker. Time is given in minutes. (AVI 5702 kb)

Parallel and perpendicular spindle positioning in asp neuroepithelial cells.

Movies of three asp dividing neuroepithelial cells expressing α-Tubulin-RFP (red in merged panel and shown separately in middle panels) to label spindle MTs and Pon-GFP (green in merged panel and shown separately in bottom panel) used as a polarity marker. Time is given in minutes. (AVI 4266 kb)

Interkinetic nuclear migration (INM) in WT neuroepithelial cells.

Movies of three WT neuroepithelial cells expressing PH-GFP to mark the membranes. Time is given in minutes. (AVI 711 kb)

Interkinetic nuclear migration (INM) in WT neuroepithelial cells.

Movie of WT neuroepithelial cell expressing PH-GFP to mark the membranes and RFP-Histone to visualize the nucleus. Notice that at time = 00:00 the apically positioned mitotic cell is already in metaphase. Time is given in hours. (AVI 4616 kb)

Interkinetic nuclear migration (INM) in WT neuroepithelial cells.

Movie of WT neuroepithelial cell expressing PH-GFP to mark the membranes and RFP-Histone to visualize the nucleus. Time is given in hours. (AVI 3253 kb)

Interkinetic nuclear migration (INM) in asp neuroepithelial cells.

Movies of three asp neuroepithelial cells expressing PH-GFP to mark the membranes. Time is given in minutes. (AVI 750 kb)

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Rujano, M., Sanchez-Pulido, L., Pennetier, C. et al. The microcephaly protein Asp regulates neuroepithelium morphogenesis by controlling the spatial distribution of myosin II. Nat Cell Biol 15, 1294–1306 (2013). https://doi.org/10.1038/ncb2858

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