QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 25 September 2007
doi: 10.1242/jcs.016626

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Rickmyre, J. L. Articles by Lee, L. A. Search for Related Content PubMed PubMed Citation Articles by Rickmyre, J. L. Articles by Lee, L. A.

The Drosophila homolog of MCPH1, a human microcephaly gene, is required for genomic stability in the early embryo

¹ Department of Cell and Developmental Biology, Vanderbilt University Medical Center, U-4200 MRBIII, 465 21st Avenue South, Nashville, TN 37232-8240, USA
² Department of Neurobiology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA
³ Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA

View larger version (60K): [in this window] [in a new window]   Fig. 1. The awol phenotype. Representative syncytial embryos (A,B) and mitotic spindles (C-K) in embryos from wild-type or awolZ1861/awolZ0978 females. (A,B) DNA staining of embryos from awol females shows arrest with condensed chromosomes and unevenly spaced nuclei (B) compared to wild type (A). (C-G) Microtubules are in green and DNA in red. (C) Asynchronous neighboring nuclei in embryo from awol female (left, interphase; right, mitosis). (D) Metaphase spindle with duplicated centrosomes in embryo from awol female shows asynchronous nuclear and centrosome cycles (duplication normally occurs in telophase). (E) Shortened, barrel-shaped spindle in embryo from awol female. (F) DNA displaced from metaphase plate is tethered by microtubules to spindle pole in embryo from awol female. (G) Wild-type spindle. (H-K) Microtubules are in green and centrosomes in red. (H-I) awol spindles with missing or ectopic centrosomes. (K) Wild-type spindle. Bars, 20 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 2. mcph1 is the awol gene. (A) The Drosophila mcph1 gene structure. Exons are represented by filled boxes, 5'- and 3'-UTRs by open boxes, and splicing events by thin lines. The gene CG13189 lies within the largest intron of mcph1. Alternative splicing produces transcript mcph1-RA or -RB. Arrows below gene or transcript names indicate direction of transcription. Positions of the point mutations in each of the three EMS-induced alleles of awol and resulting amino acid changes (numbers refer to MCPH1-B) are indicated above the mcph1 gene. Imprecise excision of P-element EY11307 (inverted triangle) generated allele mcph1Exc21 (deleted region indicated by gap). (B) Western analysis reveals trace amounts of or no MCPH1 protein in extracts of awol embryos relative to wild type (loading control: anti--tubulin). The excision allele (Exc21) of mcph1 serves as negative control. Df=Df(2R)BSC39, which removes the mcph1 genomic locus. (C) Comparison of the BRCT domain content (hatched boxes) of the two Drosophila MCPH1 isoforms (MCPH1-A and -B) and human MCPH1 protein (bottom). Positions of the amino acid changes in each of the three EMS-induced alleles of awol are indicated by asterisks. A double-sided arrow indicates the region of MCPH1-B used for antibody production.

View larger version (64K): [in this window] [in a new window]   Fig. 3. Suppression of mcph1 by Chk2 (mnk). (A-J) Representative mitotic spindles in syncytial embryos and whole-mount embryos from mcph1Z1861, mnk mcph1Z1861 and wild-type females. Bars, 20 µm. (A-F) Microtubules are in green and DNA in red; low (A,B) and high (C-F) magnification views. mcph1 embryos have awol-type (barrel-shaped, acentrosomal) spindles (A,C). awol phenotype is suppressed in mnk mcph1 embryos (B,D): note restoration of elongated spindles and attached centrosomes. Other defects are seen in mnk mcph1 embryos, such as DNA shared by two spindles (E) and DNA bridging (F, arrow). (G,H) Cellularized embryos (2-3 hours) stained for actin (green) and DNA (red). mnk mcph1 embryos reach gastrulation with irregular cell size and DNA content (G) compared to wild type (H). (I,J) DNA-stained embryos (3-4 hours). mnk mcph1 embryos (I) arrest peri-gastrulation with aberrant morphology compared to wild type (J). (K) Quantification of suppression of developmental arrest of mcph1Z1861 embryos by mnk.

View larger version (63K): [in this window] [in a new window]   Fig. 4. Chromatin bridging in mcph1 embryos. Syncytial embryos were squashed and the DNA stained. (A) Representative late anaphase-to-telophase figures (images shown at same magnification). DNA bridging and increased pole-to-pole distances are seen in squashes of mcph1Z1861/mcph1Z0978 and mnk mcph1Z1861 embryos. Bars, 10 µm. (B) Quantification of DNA bridging in mcph1Z1861/mcph1Z0978 and mnk mcph1Z1861 embryo squashes. Wild-type and mnk embryos served as controls.

View larger version (85K): [in this window] [in a new window]   Fig. 5. mcph1 larvae have intact DNA checkpoints and normal sensitivity to DNA-damaging agents. (A,B) Cell-cycle checkpoints in mcph1 larvae. Bars, 50 µm. (A) G2-M checkpoint. Eye-antennal imaginal disks were dissected from untreated (left) or irradiated (right) larvae, fixed, and stained with antibodies against phosphorylated Histone H3 (anti-PH3), a marker of mitotic cells. Lack of anti-PH3 staining post-IR indicates G2 arrest. Representative disks are shown (with at least twelve discs scored per genotype). (B) Intra-S phase checkpoint. Brains were dissected from untreated (left) or irradiated (right) larvae and labeled with BrdU. Decreased BrdU staining in brain lobes (arrows) post-IR indicates intra-S phase arrest. Representative brains are shown (with at least six brains scored per genotype). (C,D) Survival of mcph1 larvae following exposure to DNA-damaging agents. (C) Sensitivity to hydroxyurea (HU). Larvae were grown on food minus or plus HU and allowed to develop. For each genotype, the ratio of homozygous mutant to total progeny is expressed as a percentage with total number of adult flies scored shown in parentheses. (D) Sensitivity to IR. Third instar larvae were untreated or exposed to low-dose irradiation and allowed to develop. For each genotype, the ratio of eclosed adults to total pupae is expressed as a percentage with total pupae shown in parentheses.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Intact DNA-replication checkpoint and normal Cyclin B levels in mcph1 embryos. (A) Quantification of cell-cycle timing during cortical divisions of early embryogenesis. No significant differences in interphase (I) or mitosis (M) lengths were observed for mnk mcph1Z1861 embryos compared to wild-type or mnk controls, whereas shorter interphases were apparent in mei-41 embryos (cycles 12 and 13). Average times with standard deviations (error bars) are shown. Numbers of embryos scored for each genotype are shown in parentheses. (B) Western analysis using phospho-specific antibodies against Cdk1 reveals wild-type levels of pY15-Cdk1 in extracts of mnk mcph1Z1861 embryos (1-2 hours). Control grp embryos have reduced pY15-Cdk1 levels. (C) Western analysis reveals normal Cyclin B levels in mnk mcph1Z1861 embryos (1-2 hours). (D) Western analysis reveals normal GRP levels in mcph1 and mnk mcph1Z1861 embryos (1-2 hours unless otherwise indicated). Loading controls: anti--tubulin or anti-GAPDH.

View larger version (15K): [in this window] [in a new window]   Fig. 7. mcph1 cooperates with mei-41 and grp in the early embryo. (A) Mitotic spindle from a pre-cortically arrested grapesZ5170 embryo resembles awol-type spindles of mcph1 embryos. Microtubules are in green and DNA in red. Scale bar: 10 µm. (B) Quantification of mcph1-like arrest in grp embryos (2-4 hours). (C) mcph1 dominantly enhances mei-41 embryonic lethality. Introduction of one copy of mcph1Z1861 into a semi-sterile mei-41 background (mei-41RT1/mei-41D5) reduces embryonic hatch rate more than threefold. (D) Immunoblotting shows slower gel mobility of MCPH1 in mei-41RT1 or grp1 embryos (1-2 hours) relative to wild type.

View larger version (54K): [in this window] [in a new window]   Fig. 8. Defects in male mcph1 brains. Adult male brains were stained with anti-FasII antibodies to visualize mushroom body (MB) lobes and the ellipsoid body of the central complex (CC). (A) MB lobes of wild-type brains are symmetric, whereas MBs of mcph1 brains are occasionally defective with missing or diminished lobes (arrowheads). Df=Df(2R)BSC39, which removes the mcph1 genomic locus. (B) Quantification of brain defects in mcph1 males. Sample number for each genotype is indicated in parentheses (top).

View larger version (21K): [in this window] [in a new window]   Fig. 9. Proposed model of Drosophila MCPH1 function. Asterisks represent key points at which human MCPH1 reportedly functions. Our data suggest that MCPH1 cooperates with MEI-41/GRP in a Cdk1-independent manner to promote genomic integrity in embryos, possibly by controlling timing of chromosome condensation.