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First published online 28 November 2007
doi: 10.1242/dev.011072

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Matrix metalloproteinases promote motor axon fasciculation in the Drosophila embryo

¹ Department of Neurosciences, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
² Department of Biology and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, USA.

View larger version (128K): [in this window] [in a new window]   Fig. 1. Pan-neuronal misexpression of either Mmp1 or Mmp2 inhibits motor axon defasciculation. In each micrograph, two abdominal hemisegments of stage 17 dissected embryos stained with -FasII to label the motor projections are shown with anterior left and dorsal up. Below each image are schematics diagramming the observed phenotypes with motor axons in brown and muscles represented by gray boxes. In all ISNb schematics, the muscles are drawn as if transparent in order to depict both the ISN and ISNb pathways. However, note that the ISN extends in an external plane whereas the ISNb extends in an internal plane. (A,D) In wild type, ISNb axons defasciculate from the ISN at a choice point proximal to the VLM field (arrowheads), then continue to extend dorsally to innervate muscles 7, 6, 13, 12. (B,C,E,F) In elav>Mmp1 and elav>Mmp2 embryos, ISNb axons appear to defasciculate correctly from the ISN at their first choice point, but fail to defasciculate at their appropriate muscle targets, a phenotype referred to as `stall'. (G,J) In wild type, the SNa branch innervates the lateral musculature and comprises dorsal and posterior branches. The posterior branch extends posteriorly to innervate muscle 8, whereas the dorsal branch makes two stereotyped turns en route to muscles 23 and 24. (H,K) In elav>Mmp1 embryos, the dorsal branch of the SNa stalls (arrowhead) before reaching its final target. (I,L) In elav>Mmp2 embryos, the dorsal (arrowheads) and lateral branches of the SNa stall before reaching their synaptic targets. Scale bar: 15 µm.

View larger version (101K): [in this window] [in a new window]   Fig. 2. Embryonic expression profiles of Mmp1 and Mmp2. (A-E,G,H) Stage 15 wild-type embryos stained for indicated markers. (F) Stage 15 gcmP1 homozygous mutant embryo stained for Mmp2 RNA and labeled with anti-Repo. (A) Mmp1 is expressed in PNS neurons in the chordotonal organs and (B) in two cells in the ventral longitudinal muscle field (arrows). (C) Expression of Mmp2 RNA indicates that Mmp2 is expressed in a subset of CNS neurons and glia (circled in red). (D) Mmp2 RNA (green) colocalizes with anti-Wrapper (red) in midline glia. (E) Nuclear anti-Repo (brown) and cytoplasmic Mmp2 RNA (purple) in a subset of exit glia (arrows). (F) In gcmP1 embryos, which lack most glia, Mmp2 RNA is no longer expressed at the lateral edge of the CNS where the exit glia are located (arrows). (G) Anti-Islet (brown) colocalizes with Mmp2 RNA (purple) in approximately three neurons per hemisegment (arrows). The location of these neurons is consistent with an interneuron identity. (H) Anti-Hb9 (brown) does not colocalize with Mmp2 RNA (purple). Insets in G and H show high-magnification views of Mmp2 expression. Dotted white line marks the CNS boundary. Anterior is up in all panels. Dorsal is right in A,B,E,F. Scale bars: 10 µm in A,B,E,F; 20 µm in C,D,G,H.

View larger version (128K): [in this window] [in a new window]   Fig. 3. Pan-glial misexpression of Mmp1 or Mmp2 blocks motor axon defasciculation. In each micrograph, two abdominal hemisegments of stage 17 dissected embryos stained with -FasII to label the motor projections are shown with anterior left and dorsal up. Below each image are schematics of the observed phenotypes with motor axons in brown and muscles represented by gray boxes. (A,D) In wild type, axons in ISNb defasciculate from the ISN and extend dorsally to their synaptic targets. (B,C,E,F) In repo>Mmp1 and repo>Mmp2 embryos, ISNb axons fail to defasciculate appropriately and instead remain tightly bundled together, exhibiting either a `parallel bypass' (B,E); or a stronger `fusion bypass' phenotype (C,F). In fusion bypass, ISNb axons fail to separate from ISN axons at their first choice point and bypass the VLM field. (G,J) In wild type, SNa axons bifurcate into dorsal and posterior SNa branches and innervate lateral muscle targets. (H,K) repo>Mmp1 embryos exhibit mild stalls of the dorsal SNa branch (arrowheads). (I,L) repo>Mmp2 embryos display strongly truncated dorsal branches (arrowheads) and mildly stalled posterior branches. Scale bar: 15 µm.

View larger version (77K): [in this window] [in a new window]   Fig. 4. MMP LOF mutants display inappropriate ISNb defasciculation. In each micrograph, two abdominal hemisegments of stage 17 dissected embryos stained with -FasII (A-D) or -GFP (I-L) to label the motor projections, are shown with anterior left and dorsal up. Below each image are schematics of the observed phenotypes with motor axons in brown (E-H) or black (M-P) and muscles represented by gray boxes. In addition to carrying mutations in the indicated MMP genes, embryos in I-L are heterozygous for both Hb9Gal4 and UAS-mCD8GFP to visualize Hb9-positive axons with -GFP. (A,E,I,M) Wild-type embryos have tightly bundled axonal projections. (B,F,J,N) Mmp1Q112*/Mmp12 mutant embryos exhibit moderate ISNb defasciculation. (C,G,K,O) ISNb-projecting axons in Mmp2W307*/Mmp2Df embryos are loosely bundled and frequently project aberrantly within the VLM field (arrowheads). (D,H,L,P) Mmp2W307* Mmp1Q112*/Mmp2Df Mmp12 double mutants are indistinguishable from Mmp2 single mutants with frequent ectopic projections (arrowheads). Scale bar: 15 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 5. MMP LOF mutants exhibit excessive SNa defasciculation. In each micrograph, two abdominal hemisegments of stage 17 dissected embryos stained with -FasII to label the motor projections, are shown with anterior left and dorsal up. Below each image are schematics of the observed phenotypes with motor axons in brown and muscles represented by gray boxes. (A,E) In wild type, the two branches of the SNa stay tightly bundled after dividing into dorsal and posterior branches and go on to correctly innervate their lateral muscle targets. (B,F) In Mmp1Q112*/Mmp12 embryos, axons within the dorsal and posterior SNa branches project ectopically (arrowheads). (C,G) Mmp2W307*/Mmp2Df mutants display aberrant SNa defasciculation (arrowheads) phenotypes similar to those observed in Mmp1 mutants. (D,H) SNa-projecting axons in Mmp2W307* Mmp1Q112*/Mmp2Df Mmp12 double mutant embryos branch aberrantly and are less tightly bundled (arrowheads). The penetrance of SNa defasciculation observed in MMP double mutants is increased relative to either MMP single mutant. Scale bar: 15 µm.

View larger version (104K): [in this window] [in a new window]   Fig. 6. MMP catalytic activity is required for motor axon pathfinding. In each micrograph, two abdominal hemisegments of Stage 17 dissected embryos stained with -FasII to label the motor projections are shown with anterior left and dorsal up. Below each image are schematics of the observed phenotypes with motor axons in brown and muscles represented by gray boxes. (A,D) In wild type, the ISNb exhibits a highly stereotyped morphology as its axons innervate their muscle targets. (B,E) In elav>Timp embryos, ISNb axons appear loosely bundled. (C,F) In repo>Timp embryos, ISNb axons are loosely bundled and project ectopically. (G,I) elav>Mmp2E258A embryos exhibit aberrant ISNb projections with axons failing to innervate their appropriate targets. (H,J) In repo>Mmp2E258A embryos, ISNb axons are loosely bundled and often branch ectopically. Scale bar: 15 µm.

View larger version (129K): [in this window] [in a new window]   Fig. 7. The inappropriate defasciculation observed in mutants with reduced MMP activity is suppressed by increasing interaxonal adhesion. In each micrograph, two abdominal hemisegments of stage 17 dissected embryos stained with -FasII to label the motor projections are shown with anterior left and dorsal up. Below each image are schematics of the observed phenotypes with motor axons in brown and muscles represented by gray boxes. (A,D) Wild-type embryo exhibiting normal ISNb morphology. (B,E) elav>Fas2 mutant embryos have increased motor axon adhesion. (C,F) elav>Fas2, Timp embryos exhibit phenotypes consistent with weakened interaxonal adhesion relative to elav>Fas2 embryos. (G,J) Mmp2W307*/Mmp2Df mutant embryos have loosely bundled ISNb axons and ectopic branching. (H,K) The ISNb of Sema-1aP1/Sema-1aP1 mutant embryos exhibits hyperfasciculation and does not properly innervate its muscle targets. (I,L) In Sema-1aP1 Mmp2W307*/+ Mmp2Df mutant embryos, ISNb morphology resembles that of wild-type embryos. Note that these embryos have more tightly bundled nerves than do the Mmp2 homozygous mutants shown in G. Scale bar: 15 µm.

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