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The furry gene of Drosophila is important for maintaining the integrity of cellular extensions during morphogenesis

Jingli Cong, Wei Geng*, Biao He, Jingchun Liu, Jeannette Charlton and Paul N. Adler{ddagger}

Biology Department and Cancer Center, University of Virginia, Charlottesville, VA 22903, USA
* Present address: Immunology Department, Genencor International, Inc., 925 Page Mill Road, Palo Alto, CA 94304, USA



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  		Fig. 1. A map of the fry gene. A 50 kb segment is shown. The BDGP/Celera collaboration has predicted seven genes in this region. Three of these (CG6774, CG14171 and CG6780) largely comprise the fry gene. The other four are transcribed on the complementary strand and have not been studied. The relative location and size of the 28 fry exons are shown. cDNA clones have been found in two regions. One group of cDNA clones was near the 5' end of the transcript. The longest of these was about 4 kb and it included sequences from exons 3-14. A second cDNA was found as an EST (SD10447) and is located at the 3' end of the gene. Southern blot analysis and DNA sequencing were used to determine the locations of three fry mutations. The P insertion responsible for fry6 was inserted into the first intron of fry. The fry1 mutation is a frameshift mutation in exon 3. The fry2 mutation is a nonsense mutation in exon 22.

 


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  		Fig. 2. (A) A northern blot hybridized with 5' and 3' probes for both forms of the fry cDNA. The location of RNA size markers is indicated. Lane 1 contained approximately 2.5 µg of fry6/fry6 1-2 day pupal poly(A)+ RNA. Lane 2 contained 40 µg of fry6/fry6 1-2 day pupal total RNA. Lane 3 contained approximately 2.5 µg of 1-2 day Oregon R pupal poly(A)+ RNA. Lane 4 contained 40 µg of 1-2 day Oregon R total RNA. Lane 5 contained 2.5 µg of adult head poly(A)+ RNA. The filter containing lanes 1-5 was probed simultaneously with a probe for the 5' region of the fry mRNA and a probe for the approximately 500 bp rp49 mRNA as a loading control. Lane 6 contained approximately 2.5 µg of fry6/fry6 1-2 day pupal poly(A)+ RNA and lane 7 contained approximately 2.5 µg of 1-2 day Oregon R pupal poly(A)+ RNA. This filter was probed simultaneously with a probe for the 3' region of fry and with a probe for rp49 as a loading control. (B) PCR amplification of a region of the fry mRNA from a panel of Drosophila cDNAs made from a variety of developmental stages (Origene Technologies). Lane 1 is a size marker, lane 2 contains cDNA from 0-4 hour embryos, lane 3 from 4-8 hour embryos, lane 4 from 8-12 hour embryos, lane 5 from 12-24 hour embryos, lane 6 from 1st instar larvae, lane 7 from 2nd instar larvae, lane 8 from 3rd instar larvae, lane 9 from pupae, lane 10 from male heads, lane 11 from female heads, lane 12 from male bodies and lane 13 from female bodies. The fragment amplified is found in both fry-l and fry-s.

 


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  		Fig. 3. (Top) Diagrammatic representation of the Fry-L protein with the five similarity regions labeled. In parentheses is the number of amino acids in that region of the Fry protein. Shown below the Fry protein is the percentage of identical amino acids in various Fry homologs. For each of these, the number of amino acids over which the similarity to Fry is seen is given in parentheses. Note for all of the proteins that contain the 4th region, it is approximately 110 amino acids. (Bottom) Amino acid sequence for Fry, CAB42442 and AAF99910 for the first similarity domain. We call this region the Fry domain. Similar amino acids are shaded and a consensus sequence is also shown.

 


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  		Fig. 4. The wing phenotype of fry. A shows a fry1 clone seen in the SEM. This micrograph was taken of an adult wing mounted on a stud without any metal coating. The clone boundary is outlined in white. The arrows point to hairs split distally. This is shown at approximately twice the magnification of the adult cuticle in C. (B) A fry2 clone in a pupal wing stained with an anti-actin antibody to stain the developing hairs. The clone is marked by the loss of expression of the N-Myc epitope (clone cells do not stain green). The clone boundary is outlined in white. Note that only the clone cells show a fry mutant phenotype. A 2 µm size marker is shown. (C) A wing from a fry6/fry6 pharate adult. Note the strong fry mutant phenotype. (D) A confocal image of a fry6/fry6 pupal wing stained with Alexa 488 phalloidin to stain the actin cytoskeleton (this micrograph is shown at the same magnification as B).

 


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  		Fig. 5. The bristle phenotype of fry. All panels show fry6 homozygous animals. (A) Abdominal bristles from a pharate adult. Both split (arrowheads) and normal (arrows) looking bristles can be seen. Note that except for the location of the branchpoint the mutant bristles have a normal shape. (B) Several large bristles on the head of a pharate adult. These illustrate the abnormal shape seen in bristles in this region. (C) Part of the triple row from the anterior margin of the wing. Many of the stout bristles are split distally (arrowhead). (D) A confocal image of a phalloidin stained head bristle. This bristle has already started to break down actin filaments, which now appear as short segments. The bristle shows a branch (arrowhead) midway along its length. Note that actin filaments can be seen running from proximal to distal across the branch. (E) A bristle where the actin filaments become disorganized near the distal tip (arrow).

 


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  		Fig. 6. (A,B) Brightfield images of adult aristae; (C,D) Confocal projections of Alexa 488-phalloidin stained pupal aristae. (A,C) Wild-type aristae; (B,D) fry6 homozygous aristae. Large arrows point to locations of split laterals. The insert in B is a higher magnification image of a fry6 homozygous arista. Note the fine branches on the lateral. cc, the central core of the arista; l, a lateral.

 


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  		Fig. 7. A set of images taken of developing fry7 laterals over time. As the developmental rate of fry7 animals is delayed and variable, we note the first panel as 0 hours and report the number of hours since that time. Two time series are shown – one on the right and the other on the left. The arrows point to the most distal point on the lateral of interest, the arrowhead to the relevant branch-point. In the panels on the right, note the dramatic movement of the branch-point as the lateral grew. In the panels on the left, note the deformation of the lateral at 9.75 hours and its subsequent split by 16.75 hours.

 


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  		Fig. 8. Analysis of fry in wing clones. (A) A light micrograph of a region of a wing with a fry1 wing clone (outlined in black). (B) A mwh trc7 clone. (C) A trc7 clone. (D) A fry1 trc7 clone. (E) A mwh clone. (F) A mwh fry1 clone. Note the extreme phenotypes in the mwh trc7 and mwh fry1 clones. They are much stronger than either single mutant. Also note that the fry1 trc7 clone does not show a stronger phenotype than either single mutant.

 


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  		Fig. 9. Three models for the growth and splitting of laterals are shown (A-C). A lateral is shown before and after splitting near the distal tip. The expectation if subsequent growth is restricted to the distal tip, the proximal end or occurs throughout the length of the lateral, respectively. The shaded area represents material added by growth after splitting. Note that in A, the length of the arms distal to the split increases but there is no change in the distance from the branch-point to the proximal end. In B, the arms do not change length, but the distance from the branch-point to the proximal end increases. In C, growth takes place throughout the lateral. In this case we predict an increase in the length of the arms distal to the branch-point and an increase in the distance from the branch-point to the proximal end. Our observations on fry arista laterals are consistent with C.

 

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