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 October 6, 2003
doi: 10.1242/10.1242/dev.00792

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 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 Lee, A.
Right arrow Articles by Gao, F.-B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, A.
Right arrow Articles by Gao, F.-B.
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?

Control of dendritic development by the Drosophila fragile X-related gene involves the small GTPase Rac1

Alan Lee1, Wenjun Li1,2, Kanyan Xu1,2, Brigitte A. Bogert1,2, Kimmy Su1 and Fen-Biao Gao1,2,*

1 Gladstone Institute of Neurological Disease, University of California, San Francisco, San Francisco, CA 94141-9100, USA
2 Neuroscience Graduate Program, University of California, San Francisco, San Francisco, CA 94141-9100, USA



View larger version (22K):

[in a new window]
  		Fig. 1. Generation of Fmr1 mutant flies. (A) A P-element inserted in the first intron of the Fmr1 gene can be overexpressed because of the Gal4-binding sites in the P-element. (B) Western blot analysis indicated that Fmr1, an 80 kDa protein with multiple isoforms, is not expressed in mutant lines Fmr11, Fmr12 and Fmr14. (C) Fmr1 contains several highly conserved domains, including the FMR1/FXR-interacting domain, KH domains and the ribosomal-association domain. EMS-induced mutations have been identified. For example, a point mutation in the Fmr14 allele changes amino acid 289 to a stop codon, and a small deletion in the Fmr11 allele causes a frame-shift in the N terminus after the amino acid 126.

 


View larger version (82K):

[in a new window]
  		Fig. 2. Subcellular localization of Fmr1 in DA neurons in Drosophila larvae. (A) Schematic representation of all the neurons in the dorsal cluster of an abdominal hemisegment. des, dorsal external sensory neurons; dda, dorsal dendritic arborization neuron;. dbd, dorsal bipolar dendritic neuron. Only the dda and dbd neurons are labeled by GFP using Gal4 109(2)80. (B) Cytoplasmic localization of Fmr1-GFP fusion protein in a ddaF neuron is indicated by the arrow. The expression of Fmr1-GFP was driven by Gal4 109(2)80. Scale bar: 10 µm. (C) The Fmr1-GFP signal in dendrites (arrow) is relatively weak compared with that in the cell body. This image was enhanced using Photoshop to demonstrate the localization of Fmr1-GFP in dotted structures in dendrites of dorsal cluster DA neurons. (D) UAS-GFP driven by Gal4 109(2)80 in these DA neurons indicates the dendritic branching patterns. (E) GFP-labeled DA neurons in a wild-type third instar larva are indicated by arrows. (F) Antibody staining of the same DA neurons as in E demonstrates the cytoplasmic localization of endogenous Fmr1. The arrowhead indicates the proximal segments of dendrites. The arrow indicates a muscle fiber that was also labeled by Fmr1-specific monoclonal antibody. (G) UAS-GFP-labeled DA neurons in a Fmr1 mutant third instar larva are indicated by arrows. (H) Antibody staining of the same DA neurons in Panel G demonstrates the absence of Fmr1 positive signals in Fmr1 mutants.

 


View larger version (45K):

[in a new window]
  		Fig. 3. Morphological alterations in DA neurons caused by Fmr1 mutations. (A,B) Ventral DA neurons in the A5 segment of a wild-type larva (w1118) (A) and a Fmr14 mutant larva (B) were labeled by UAS-mCD8::GFP. The images were taken from live animals, and all the dendritic processes could be visualized. (C) All ends in a specific area between the two ventral clusters of DA neurons in the A5 segment were counted to determine the number of dendritic processes per 1000 µm2 (n=38 for wild-type, n=35 for mutants, ***P<0.001). All the values are mean±s.e.m. To rescue the dendritic phenotype in Fmr1 mutants, a chromosome containing a 14 kb fragment that spans the Fmr1 transcriptional unit (Dockendorff et al., 2002Go) was introduced into the Fmr14 mutant background. (D) The distribution of individual larvae with different numbers of dendritic ends illustrates the differences among individual animals of the same genotype and between wild-type and mutant larvae. Scale bars: 40 µm.

 


View larger version (151K):

[in a new window]
  		Fig. 4. Dendritic defects caused by overexpression of Fmr1 in DA neurons of wild-type larvae. (A) Ventral DA neurons in each hemisegment elaborate their dendrites underneath the ventral epidermis. The dendritic field near the cell bodies of ventral cluster DA neurons in a wild-type larva is shown. (B) Overexpression of Fmr1 in ventral DA neurons reduced the number of dendritic processes. (C) All dendritic processes in a specific area in the A5 segment were counted, and the average numbers for wild-type and Fmr1 mutant larvae are presented (n=25, ***P<0.001). (D) Dorsal cluster DA neurons in the A5 segment of a wild-type larva elaborated higher-order dendritic processes near the dorsal midline. (E) Fmr1 overexpression reduced the number of terminal dendritic processes near the dorsal midline. Scale bars: 40 µm. The arrows in D,E indicate the dorsal midline.

 


View larger version (40K):

[in a new window]
  		Fig. 5. Rac1 mRNA is present in Fmr1-mRNP complexes in vivo. DNA fragments obtained from PCR were analyzed by electrophoresis in a 2% agarose gel. DNA ladders were run on both sides of the gel. Total RNA: DNA fragments derived from RT-PCR reactions with total RNA isolated from third instar larvae. Primers specific for the voltage-gated K+ channel molecule Hyperkinetic gave rise to DNA fragments of 0.3 kb. The band for {alpha}-tubulin is 1.0 kb, and the band for Rac1 is 0.2 kb. WT: DNA fragments derived from RT-PCR reactions with RNAs isolated from the Fmr1-mRNP complexes immunoprecipitated from wild-type third instar larvae. Fmr14: DNA fragments derived from RT-PCR reactions with RNAs isolated from the Fmr1-mRNP complexes immunoprecipitated from Fmr1 mutant third instar larvae.

 


View larger version (125K):

[in a new window]
  		Fig. 6. Rac1 is required for dendritic branching of DA neurons in Drosophila larvae. (A) A wild-type ddaC neuron that elaborates extensive dendritic arbors. (B,C) Rac1 mutant ddaC neurons with fewer dendritic branches. (D) An enlarged image of the area indicated by a square in A. (E) An enlarged image of the area indicated by a square in C. (F) Statistical analysis of the numbers of terminal dendritic branches for wild-type (n=15) and Rac1 mutant ddaC neurons (n=7) (P<0.01). Scale bars: 50 µm.

 


View larger version (121K):

[in a new window]
  		Fig. 7. Expression of Rac1 increases dendritic branching of DA neurons in Drosophila larvae. (A) Dendritic branching pattern of dorsal cluster DA neurons in wild-type third instar larvae. (B) Increased dendritic branching when Rac1 is overexpressed in DA neurons. (C,D) Enlarged images of dendrites in A,B, respectively. (E) Reduced dendritic branching when Fmr1 is overexpressed in dorsal cluster DA neurons. (F) Reduced dendritic branching caused by Fmr1 overexpression (E) can be partially reversed by expression of Rac1. Scale bars: 40 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 2003