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

First published online 13 September 2006
doi: 10.1242/dev.02580

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend 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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Meyer, F. Articles by Aberle, H. Search for Related Content PubMed PubMed Citation Articles by Meyer, F. Articles by Aberle, H. Social Bookmarking                         What's this?

At the next stop sign turn right: the metalloprotease Tolloid-related 1 controls defasciculation of motor axons in Drosophila

Max-Planck-Institute for Developmental Biology, Department III/Genetics, Spemannstrasse 35, 72076 Tübingen, Germany.

View larger version (88K): [in a new window]   Fig. 1. Neuromuscular terminals are missing and mislocalized in piranha mutant third instar larvae. Arrows illustrate the normal localization of NMJs on muscles of wild-type larvae. (A) Schematic diagram of the innervation pattern of the somatic musculature of an abdominal hemisegment viewed from the exterior. The approximate branching pattern of the five motor nerves and the innervation sites are superimposed onto the muscle pattern. The approximate locations of motoneurons in the ventral nerve cord are indicated. (B-D) Innervation defects on dorsalmost muscles. (B) Schematic diagram and (C) confocal micrograph of NMJs (bright green) on muscle pairs 1/9 and 2/10 in undissected wild-type larvae as seen with the CD8-GFP-Sh marker (exterior view). (D) NMJs are missing on muscle 1 and reduced to a remnant on muscle 9 in this piranhaD427/piranhaK788 mutant hemisegment. (E-G) Innervation defects on ventral muscles. (E) Schematic diagram and (F) confocal micrograph depicting the locations of NMJs on ventral internal muscles 12, 13, 6 and 7 in an undissected wild-type larva expressing CD8-GFP-Sh. (G) In this piranhaD427/piranhaK788 mutant hemisegment, NMJs are missing on muscle 13 and rudimentary in the cleft of muscles 6/7. Muscle 12 displays innervation defects as well. Asterisks indicate NMJs and muscles outside of the intended focal plane. (H-M) Confocal images of the ventral innervation pattern of a dissected wild-type (H-J) and piranhaD427/piranhaK788 mutant larva (K-M) viewed from the interior. NMJs on muscles 12, 13, 6 and 7 are visualized with CD8-GFP-Sh (H,K) and anti-Dlg (I,L). Endogenous Dlg staining completely overlaps with transgenic CD8-GFP-Sh (J,M). Note the altered innervation pattern of muscles 12 and 13 and the lack of NMJs in the cleft between muscles 6 and 7 in the mutant animal (arrows in K-M). Dorsal is up and anterior is left in all figures. AC, anterior commissure; PC, posterior commissure.

View larger version (94K): [in a new window]   Fig. 2. Motor axons are misguided in piranhaD427/piranhaK788 mutant larvae. (A-F) Confocal micrographs of the ISNb innervating ventral muscles 12, 13, 6 and 7 in dissected wild-type (A-C) and piranha mutant (D-F) larvae. Muscles and postsynaptic terminals are visualized with CD8-GFP-Sh (A,D), motor axons and presynaptic endings are stained with anti-Fasciclin II (B,E). The ISNb normally innervates muscle 12 on its ventral side via a ventrodorsal projection (arrowheads in B,C). In a piranha mutant hemisegment, muscles 12, 13 and 6 are innervated at ectopic sites by a dorsoventral nerve projection (arrowheads in E,F). Muscles 6 and 7 remain uninnervated (arrows in D-F). Arrows mark the cleft between muscles 6 and 7. Asterisks indicate type II boutons. (G-L) Transgenic labeling of motor axons reveals guidance defects in living piranhaD427/piranhaK788 mutant larvae (exterior views). In addition to CD8-GFP-Sh, larvae express dsRed2 in all motoneurons using OK371-Gal4. (G,J) Schematic diagram (G) and confocal micrograph (J) of the hemisegmental nerve defasciculating into five nerve branches at the ventral choice point of a wild-type larva. (H,K) Schematic diagram (H) and confocal micrograph (K) of the defasciculation defects in a piranha mutant larva. The ISNb failed to branch into the ventral region and migrated in a parallel pathway along the ISN. The ISN, ISNb and SNa pathways are visible as separate nerve bundles (arrow). An axon branching out of the misguided ISNb innervates muscle 13 at an ectopic position (arrowhead). (I,L) Schematic diagram (I) and confocal micrograph (L) of the ventral muscle field of a piranha mutant larva. The SNa stays attached to the SNc and defasciculates too late (arrow). Asterisks in (J-L) indicate nerves innervating neighboring hemisegments.

View larger version (136K): [in a new window]   Fig. 3. Axon guidance defects in piranha mutants arise during embryonic development and affect all motor axon pathways. (A-D) Micrographs of motor axons in three consecutive hemisegments in dissected wild-type (A,C) and piranhaD427/piranhaK788 mutant (B,D) embryos at stage 17 stained with anti-Fas II antibodies. (A) In the dorsal region of a wild-type embryo, the ISN has reached its terminal branch point at muscle pair 1/9 in all three hemisegments, and all three branch points are well developed (arrowheads 1-3). (B) The ISN fails to reach its final branch point properly (arrow) in piranha mutants, and most branch points appear underdeveloped (arrowheads 1-3). (C) Lateral and ventral body wall region of a wild-type embryo. The SNa bifurcates into a dorsal branch (arrow) and posterior branch. The ISNb (arrowheads) forms a clearly visible projection innervating muscles 12, 13, 6 and 7. (D) In piranha mutants, a bifurcated dorsal branch of the SNa is often observed (arrowheads). The ISNb fails to innervate muscles 12, 13, 6 and 7. In the left segment, the ISNb remains fused to the ISN and branches off at muscle 4 (arrow). In the middle and right segments, a split bypass occurs, with the ISNb temporarily detaching from the ISN (right) or fusing to different nerve tracts (middle).

View larger version (67K): [in a new window]   Fig. 4. Positional cloning of piranha. (A) Genomic organization of the piranha (tolloid-related 1, tlr1) locus. The 3' end of the tlr1 gene is less than 1 kb away from the 5' end of its paralog tolloid (tld), limiting the 5' regulatory region of tld to a minimum. (B) Domain structure of Drosophila Tlr1, Tld and their mouse homologs. The EMS-induced point mutations in the four piranha (tlr1) alleles are indicated (see text). (C-F) In situ hybridization using tlr1 antisense probes. tlr1 is expressed in the dorsal blastoderm at stage 5 (C), in progenitor cells of the visceral mesoderm at stage 10 (D), in somatic muscles at stage 13 (E) and in cells of the ventral nerve cord and ring gland (arrow) at stage 16 (F).

View larger version (138K): [in a new window]   Fig. 5. The tlr1 mutant phenotype can be rescued by expressing wild-type Tlr1 in various tissues. (A-F) Confocal micrographs of NMJs on ventral muscles 12, 13, 6 and 7 stained with CD8-GFP-Sh in undissected third instar larvae. Arrows indicate NMJs on muscles 12, 13, 6 and 7. (A) Wild type. (B) tlr1D427/tlr1K788 mutant larva carrying Heat shock-Gal4 and UAS-tlr1 transgenes. When no heat shock is applied, the neuromuscular pattern is disrupted. Muscles 12 and 13 are innervated at wrong positions, and the cleft between muscles 6 and 7 is only marginally innervated (arrows). (C) Application of a heat shock during mid-embryogenesis completely rescues the innervation errors on all muscles. (D) Ventral muscle field of a tlr1D427/tlr1K788 mutant larva expressing Tlr1 in all somatic muscles using G14-Gal4. The innervation pattern appears wild-type. (E) Expression of Tlr1 in all postmitotic neurons using Elav-Gal4 similarly rescues the innervation errors in tlr1D427/tlr1K788 mutants. (F) The neuromuscular pattern is not restored in tlr1D427/tlr1K788 mutants expressing Tlr1 only in cholinergic neurons using Cha-Gal4. NMJs on muscles 12 and 13 are mislocalized (arrows). Asterisks indicate NMJs located on muscles that are outside the intended focal plane.

View larger version (65K): [in a new window]   Fig. 6. Tolloid cannot functionally replace Tlr1. (A-F) Confocal images of the innervation patterns of ventral internal muscles in intact third instar larvae stained with CD8-GFP-Sh. Arrows indicate the locations of NMJs on muscles 12, 13, 6 and 7. (A) In a tlr1D427/tlr1K788 mutant animal, NMJs are mislocalized on muscle 12 and reduced to a remnant in the cleft of muscles 6 and 7. (B) All terminals are at their correct location and structured normally in a tolloid (tld10E95/tld9B66) mutant larva. (C) The neuromuscular innervation pattern appears wild-type in kuzbanian (kuze29/kuzK01403) mutants. (D) Overexpression of Tlr1 in all neurons using Elav-Gal4 rescues the tlr1 mutant phenotype. (E) Overexpression of Tld with Elav-Gal4 does not rescue the NMJ pattern in tlr1 mutants. NMJs on muscles 12 and 13 are mislocalized in this hemisegment. (F) Overexpression of Kuzbanian in all neurons does not restore the innervation defects in tlr1 mutants. NMJs are mislocalized on muscles 12 and almost absent on muscles 13, 6 and 7. Asterisks indicate NMJs located on muscles that are outside of the intended focal plane.

View larger version (134K): [in a new window]   Fig. 7. Innervation defects are strongly enhanced in tlr1,side double mutants. Arrows indicate missing or mislocalized NMJs on muscles 12, 13, 6 and 7. Asterisks mark NMJs outside of the intended focal plane. (A-C) Confocal images of tlr1D427 (A) and sideC137/sideI1563 (B) single and tlr1D427,sideC137/tlr1K788,sideC137 (C) double mutant third instar larvae stained with CD8-GFP-Sh. Double mutant larvae (C) show a strong enhancement of the innervation phenotype and lack almost all NMJs on ventral muscles, only muscle 12 is innervated at an unusual dorsal-posterior position in this example. (A'-C') Confocal images of dissected tlr1D427/tlr1K788 (A') and sideC137/sideI1563 (B') single and tlr1D427,sideC137/tlr1K788,sideC137 (C') double mutant third instar larvae stained with CD8-GFP-Sh (green) and anti-Fas II antibodies (red). In this double mutant hemisegment (C'), all NMJs are missing on ventral muscles, including NMJs formed by type II boutons. (A"-C") Schematic representation of the quantitative evaluation of the larval innervation defects as presented in Table 1. The frequency of innervation errors for a respective muscle in wild type was subtracted from the frequency observed in mutants. Misinnervation frequencies were then transformed into a color code, as depicted in (A"). Muscles 4 and 25 were not evaluated. In tlr1 mutants (A"), the misinnervation phenotype is weaker than in side mutants (B"), and creation of a double mutant aggravates the innervation defects in ventral, lateral and dorsal body wall regions (C").

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter