spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 28 February 2006
doi: 10.1242/jcs.02834

Journal of Cell Science 119, 1144-1153 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in JCS
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chang, C.-J.
Right arrow Articles by Carmena, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chang, C.-J.
Right arrow Articles by Carmena, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Drosophila Incenp is required for cytokinesis and asymmetric cell division during development of the nervous system

Chih-Jui Chang*, Sarah Goulding{ddagger}, Richard R. Adams§, William C. Earnshaw and Mar Carmena

Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Kings Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK


Figure 1
View larger version (62K):

[in a new window]
  		Fig. 1. incenpP(EP)2340 individuals show defects in chromosome segregation and cytokinesis. (A) Diagram of the Incenp genomic region showing insertion site of the P-element into the third exon of the Incenp gene in incenpP(EP)2340. Black lines on either side of the P-element represent the genomic DNA fragments cloned by plasmid rescue and sequenced. Red boxes indicate the position of primers used for the PCR characterization of imprecise excision lines. Diagram of the Incenp transcript below shows the position of the point mutation (C to T) in incenpEC3747. Below, diagrams of the predicted products of both Incenp mutants. (B) Northern blot, showing total RNA from wild-type and both Incenp mutants. Arrow points to truncated transcript present in heterozygous incenpP(EP)2340/+ individuals. (C) Western blot analysis of wild-type embryos (WT), mutant embryos carrying a deficiency for the Incenp gene (DF), incenp3747 homozygous embryos (3747) and incenpP(EP)2340 homozygous embryos [3747, [P(EP)]. Arrow points to wild-type Incenp protein. Lower panel shows {alpha}-tubulin loading control. (D) Phenotype of incenpP(EP)2340/+ in male meiosis. (Left) Wild-type onion-stage cyst; arrow points to individual spermatid with equal-size nucleus and mitochondrial derivative (Nebenkern). (Right) Same stage in incenpP(EP)2340/+ males; notice large Nebenkerns indicative of defective cytokinesis (arrowhead), as well as variable-size nuclei indicative of problems in chromosome segregation (arrow).

 

Figure 2
View larger version (121K):

[in a new window]
  		Fig. 2. Phenotypic analysis of the new genetic interactors of incenpP(EP)2340. (A-B) Normal eye and wing morphology in control (w1118) strain. (C-D) Representative examples of eye and wing phenotypes observed in trans-heterozygous combinations of EMS-induced mutations over incenpP(EP)2340: (C) Reduced eyes in trans-heterozygous EC3959/incenpP(EP)2340. (D) Irregular nicked wing-shape in trans-heterozygous EC2519/incenpP(EP)2340 fly. (E,F) Defects in the primary spermatocytes in trans-heterozygous males as a result of abnormalities in the gonial mitoses. (E) Variation in the nuclear size in EC2519/incenpP(EP)2340 is an indication of defective chromokinesis. Arrows point to micronuclei, arrowhead to polyploid nucleus. (F) The presence of giant primary spermatocytes (arrows) in EC2394/incenpP(EP)2340 could be indicative of a defect in cytokinesis in gonial mitosis. (G) Wild-type primary spermatocyte cysts. (H,I) Abnormal (H) meiotic spindles and (I) tetrapolar spindles (arrows) in EC2394/incenpP(EP)2340. (J) Wild-type telophase I spindles. Dotted lines in H-J delimit the meiotic cyst. Bar, 10 µm.

 

Figure 3
View larger version (70K):

[in a new window]
  		Fig. 3. Peripheral neuron system morphology incenpEC3747 embryos. (A-D) Examples of 20-hour embryos stained for the neural marker Mab22C10. Small blue arrows indicate the position of the lateral cluster of chordotonal neurons. (A) incenpEC3747/CyO KrGFP embryo. The wild-type PNS pattern is visible. (B-D) Homozygous incenpEC3747 embryos. (B and C) Small blue arrows show examples of neural loss from the lateral cluster. (D) Green arrows show a lack of organization of neuron clusters and reduced number of neurons. In these images anterior is left, dorsal is top. The views in C and D are more ventral than those of A and B.

 

Figure 4
View larger version (83K):

[in a new window]
  		Fig. 4. Drosophila Incenp becomes undetectable at stage 13 of embryonic development in incenpEC3747. Ventral view of stage-13 embryos. (A-A") Wild-type embryos, showing mitotic cells staining positive for (A') Drosophila Incenp and (A") phosphorylated histone H3. (B-B") Homozygous incenpEC3747 embryos at the same stage show no (B') Drosophila Incenp or (B") phosphorylated histone H3. Insets in A and B show the DNA staining (DAPI). Notice that the embryos have developed normally up to this stage. In the merged figures, DAPI is shown in blue, phosphorylated histone H3 in red, Drosophila Incenp in green.

 

Figure 5
View larger version (85K):

[in a new window]
  		Fig. 5. incenpEC3747enlarged CNS cells lack Drosophila Incenp and phosphorylated histone H3. (A-A'") Mitosis in a wild-type CNS cell showing Drosophila Incenp localized to the centromere at prometaphase. Histone H3 is phosphorylated in the mitotic chromosomes. (B-B'") Enlarged cells in the CNS of IncenpEC3747 embryos do not show any Drosophila Incenp phosphorylated histone H3 staining. In the merged figures, DAPI is shown in blue, phosphorylated histone H3 in red, Drosophila Incenp in green. Enlarged CNS cells in incenpEC3747 embryos are polyploid and show multiple centrosomes. (C-C'") Wild-type cell with Drosophila Incenp localized to centromeres at metaphase (arrowhead) and in the midzone at telophase (arrow). (D-E'") Enlarged CNS cells are polyploid and show either and abnormal accumulation of {gamma}-tubulin in centrosomes or multiple centrosomes. The two centrosomal masses in D-D'" have not separated properly in this cell. Multiple {gamma}-tubulin spots in E-E'". In the merged figures, DAPI is shown in blue, {gamma}-tubulin in red, Drosophila Incenp in green. Bar, 10 µm.

 

Figure 6
View larger version (65K):

[in a new window]
  		Fig. 6. Localization of the cell-fate determinant Prospero in mitosis of wild-type and incenpEC3747 embryos. (A-C) Localization of Prospero in mitosis of wild-type CNS cells. (A) In early prophase, Drosophila Incenp shows general association with chromatin, whereas Prospero localizes in discrete spots associated with chromatin. (B) By early prometaphase, both proteins become closer although they do not colocalise totally. (C) In metaphase, Drosophila Incenp remains associated with centromeres, whereas Prospero is localized in a crescent in the basal cell cortex. (D-D') In incenpEC3747 homozygous embryos, we observed enlarged polyploidy cells depleted of Drosophila Incenp in which Prospero localization is abnormal. All images are projections of series of deconvolved images. Bars, 5 µm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006