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Regulation of CNS and motor axon guidance in Drosophila by the receptor tyrosine phosphatase DPTP52F

Benno Schindelholz1,*, Matthias Knirr1,{ddagger}, Rahul Warrior2 and Kai Zinn1,§

1 Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA
2 Department of Biology, University of Southern California, 835 W 37th Street, Los Angeles, CA 90089, USA
* Present address: The Genetics Company, Winterthurstr. 190, 8057 Zürich, Switzerland
{ddagger} Present address: Friedrich-Miescher-Labor der Max-Planck-Gesellschaft, Spemannstr. 37, D-72070 Tübingen, Germany



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  		Fig. 1. The Ptp52F gene. (A) Map of the deficiencies used for the EMS lethal screen. The position of the Ptp52F gene is indicated by the shaded bar. (B) Exon maps of Ptp52F and the adjacent DLis1 genes; distal (toward the telomere) is towards the left, so the orientation is flipped relative to A. (C) Sequence of the DPTP52F preprotein. Indicated sequences are: putative signal sequence, thin underline; FN3 repeats, thick underlines; putative transmembrane domain, double underline; PTP domain, broken underline; bold, highly conserved PTP domain residues; letters in shaded bars, amino acid changes produced by mutations. (D) Lineup of the DPTP52F FN3 repeats with two FN3 repeats from DPTP10D and the seventh FN3 repeat in human fibronectin. Shaded bars, conserved residues. Consensus residues indicated in bold.

 


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  		Fig. 2. Expression of DPTP52F protein. Whole-mount embryos were stained with mAb 13B8 using HRP immunohistochemistry. (A) A ventral view of a gastrulating embryo, showing DPTP52F at the edges of the ventral furrow (arrow). At this stage, DPTP52F is on the membranes of all cells in the embryo at lower levels. (B) A side view (anterior towards the left) of a stage 15 embryo. The ventral nerve cord (arrow) expresses DPTP52F. (C) Two segments of a dissected CNS from a stage 15 wild-type embryo. DPTP52F is expressed on cell bodies; axon tracts (arrow) are lighter. (D) A dissected CNS from a stage 15 Ptp52F18.3 embryo. No staining is observed.

 


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  		Fig. 3. CNS and motor axon phenotypes produced by RNAi. Dissected late stage 16 embryos stained with mAb 1D4. Column 1 (A,E,I), wild-type. Column 2 (B,F,J), embryos injected with injection buffer. Column 3 (C,G,K), embryos injected with Dlar dsRNA. Column 4 (D,H,L), embryos injected with Ptp52F dsRNA. Row 1 (A-D), the CNS (anterior up); note the 3 straight longitudinal bundles on each side of the midline in A-C. Arrow in D indicates a break in the outer 1D4 bundle; arrowhead indicates abnormal fasciculation of the middle and outer bundles. Row 2 (E-H), ISNb (anterior towards the left); note the 3 characteristic synaptic branches in E,F,H; muscles are numbered in E. Arrow in G, bypass ISNb; arrowhead, ISN; asterisk, SNa. Row 3 (I-L), SNa; note the characteristic bifurcation in I-K, indicated by arrow in (I); muscle 12 is also labeled. Asterisk in L, approximate position of missing bifurcation.

 


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  		Fig. 4. CNS phenotypes in Ptp52F and double mutants. Dissected late stage 16 embryos stained with mAb 1D4. (A) wild-type. (B) Ptp52F18.3. (C) Ptp52F18.3/Df(2R)JP4. The bundles are wavy and disorganized in B,C, and the outer bundle is sometimes missing (bracket). (B,C,E) Abnormal fasciculation of the middle and outer bundles (arrowheads). (D) Dlar5.5Ptp52F18.3. Bundle morphology in this double mutant reverts to wild type (compare with A-C). (E) Ptp10D1,Ptp52F18.3. (F) Ptp52F18.3, Ptp69D1/Df(3L)8ex25. 1D4-positive bundles in these two double mutants are more disorganized than in B,C, and the middle bundle is strongly affected (asterisks). Arrow, complete connective break.

 


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  		Fig. 5. The pCC growth cone stalls in Ptp52F mutants. Dissected embryos stained with mAb 1D4. (A) wild-type, late stage 12. aCC growth cones (a) have just begun to turn outwards. The pCC growth cone has already grown onto the 1D4-positive LG5 cell (bracket) in the hemisegments on the right (indicated by parallel lines flanking the pCC axon connection to LG5). (B) Ptp52F18.3, early stage 13. The aCC growth cone (a) has already turned posteriorly and then begun to extend away from the CNS, but the pCC growth cones (p) have still not reached LG5. (C) Wild-type, early stage 13. The aCC growth cone (a) is at the same position as the one indicated in B. A continuous longitudinal pathway has formed. pCC axons are indicated by flanking parallel lines. (D) Ptp52F18.3, late stage 13. The aCC axons (a) have already extended beyond the edges of the panel, but there are still gaps in the longitudinal pathways on the left side (arrows) just anterior to the pCC cell bodies.

 


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  		Fig. 6. SNa phenotypes in Ptp52F mutants. Dissected late stage 16 embryos stained with mAb 1D4. (A) Wild type. The SNa bifurcation (arrow) and muscle 12 are indicated. (B-D) Ptp52F18.3. There is an extra branch in B (arrow). The anterior branch is missing in C. Most SNa axons appear to stall near the bifurcation point in D (arrow).

 


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  		Fig. 7. ISNb and ISN phenotypes in Ptp52F and double mutants. Dissected late stage 16 embryos stained with mAb 1D4. (A) Ptp52F18.3. The ISNb has a normal appearance. Muscles are labeled. (B) Ptp10D1, Ptp52F8.10.3.. The left ISNb has stalled at the proximal edge of muscle 13 (arrow) without making a synaptic branch. The right ISNb is also truncated. The distal portion of the ISN is shown in C-F. Terminal arbors are indicated by arrowheads in C,E; the large tracheal branch just distal to the second branchpoint is indicated by asterisks in C-F. (C) Ptp52F8.10.3. (D) Ptp69D1/Df(3L)8ex25. The ISN has a normal appearance in these single mutants. (E) Ptp10D1, Ptp52F8.10.3.. Terminal arbors are reduced in this double mutant. (F) Ptp52F8.10.3, Ptp69D1/Df(3L)8ex25. ISNs stall at the second branchpoint position (arrow), proximal to the tracheal branch.

 

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© The Company of Biologists Ltd 2001