Drosophila MMP2 regulates the matrix molecule faulty attraction (Frac) to promote motor axon targeting in Drosophila

Abstract

Matrix metalloproteinases (MMPs) are widely hypothesized to regulate signaling events through processing of extracellular matrix (ECM) molecules. We previously demonstrated that membrane-associated Mmp2 is expressed in exit glia and contributes to motor axon targeting. To identify possible substrates, we undertook a yeast interaction screen for Mmp2-binding proteins and identified the novel ECM protein faulty attraction (Frac). Frac encodes a multidomain extracellular protein rich in epidermal growth factor (EGF) and calcium-binding EGF domains, related to the vertebrate Fibrillin and Fibulin gene families. It is expressed in mesodermal domains flanking Mmp2-positive glia. The juxtaposition of Mmp2 and Frac proteins raises the possibility that Frac is a proteolytic target of Mmp2. Consistent with this hypothesis, levels of full-length Frac are increased in Mmp2 loss-of-function (LOF) and decreased in Mmp2 gain-of-function (GOF) embryos, indicating that Frac cleavage is Mmp2 dependent. To test whether frac is necessary for axon targeting, we characterized guidance in frac LOF mutants. Motor axons in frac LOF embryos are loosely associated and project ectopically, a phenotype essentially equivalent to that of Mmp2 LOF. The phenotypic similarity between enzyme and substrate mutants argues that Mmp2 activates Frac. In addition, Mmp2 overexpression pathfinding phenotypes depend on frac activity, indicating that Mmp2 is genetically upstream of frac. Last, overexpression experiments suggest that Frac is unlikely to have intrinsic signaling activity, raising the possibility that an Mmp2-generated Frac fragment acts as a guidance cue cofactor. Indeed, we present genetic evidence that Frac regulates a non-canonical LIM kinase 1-dependent bone morphogenetic protein signaling pathway in motoneurons necessary for axon pathfinding during embryogenesis.

Figure 1.

frac gene structure and allele generation. A, Domain structure of Drosophila Frac and Homo sapiens Fibrillin-1, Fibulin 1, and Fibulin 7. Two Fibulins were included to account for the variation between family members. Domains pictured include EGF-like repeats (green), cbEGF (orange), CCP/SUSHI (blue), hyalin (yellow pentagon), TB (gray hexagon), and fibulin (gray circles). B, Schematic of the frac gene region and span of frac LOF alleles. Exons are represented by rectangular black boxes and introns by thin lines. Position of Minos insertion is displayed.

Figure 2.

frac RNA and Frac protein are expressed in the mesoderm and muscle attachment sites during embryogenesis. A, B, Stage 13 and stage 15 wild-type embryos hybridized with antisense frac RNA probe. C–E, Stage 14 (C), stage 15 (D), and stage 17 (E) wild-type embryos labeled with anti-Frac. F–I, Wild-type embryos colabeled with Frac in green and indicated antibodies in red. F, At stage 15, Frac colocalizes with the muscle marker MHC (red). G, During late stage 17, Frac protein colocalizes with Kakapo/Shortstop, marking muscle attachment sites (red). H, At stage 14, Frac protein is directly adjacent to the VNC from which axons, marked with 1D4 (red), are extending (dashed white lines mark the border of the VNC). I, Frac borders the Mmp2-expressing exit glia (arrows), a subset of glia marked by anti-Repo (red). J, repoGAL4>actinGFP embryos have glial processes marked by GFP (red) adjacent to Frac protein (green) expression in the periphery (arrows). A–E, Anterior is up; F–J, anterior is left, and midline is at the bottom. Scale bars, 100 μm.

Figure 3.

Frac protein processing is Mmp2 dependent. A, Western blot with Frac antibody. Bottom shows GAPDH loading control. Full-length Frac is detected in wild-type embryo extracts at 170 and 250 kDa. Nonspecific bands are indicated by brackets in the frac deficiency column. In Mmp2 GOF embryos, cleavage products are present at 17, 30, and 33 kDa (arrowheads). B, Quantification of the full-length Frac bands (see box in A) in relation to the density of the 22 kDa background band (significance calculated using ANOVA; n = 9 blots; *p < 0.05, **p < 0.01). C, D, Stage 14 wild-type (C) and tub>Mmp2 (D) embryos labeled with Frac antibody. Anterior is up. Scale bar, 50 μm. E, Quantification of Frac protein expression in indicated genotypes. Stage 14 embryos were dissected and scored blindly on a scale of 0–3 for strength of Frac protein expression. The number of embryos analyzed is indicated below the histogram. Each genotype was individually compared with wild type using the Student's t test (***p < 0.001).

Figure 4.

A, B, frac LOF mutants display misprojections and defasciculation defects similar to Mmp2 mutants. Stage 14 wild-type (A) and frac^Δ1 (B) embryos labeled with Frac antibody. C, D, Lateral view of stage 16 embryos marked with anti-FasII, labeling all motor axon projections, and anti-GFP. In addition to the marked genotypes, embryos carry viking::GFP to visualize the cadherin-positive basement membrane. Wild-type (C) and frac^Δ1 (D) homozygous mutant embryos all have Collagen IV (green) expression at the basement membrane surrounding the ventral nerve cord (red) and midgut. E, F, MHC antibody was used to visualize the segmentally repeated pattern of muscles in each embryo. In wild-type (E) and frac^Δ1 (F) homozygous mutant embryos, muscles are properly formed and attached to the epidermis at muscle attachment sites (arrow). G–V, In each micrograph, two abdominal hemisegments of stage 17 dissected embryos stained with anti-FasII to label the ISNb (G–J) or SNa (O–R) motor projections are shown. Below each image are schematics of the branching patterns with the motors axons in brown and the muscles represented by gray boxes. G, K, In wild type, the ISNb defasciculates from the main branch of the ISN and then innervates four muscle clefts. H, I, L, M, frac^Δ1 and frac^Δ2 homozygous mutants display frayed ISNb axons and axons that separate at incorrect sites (arrows). I, M, In wild type, the SNa extends dorsally to lateral muscles and then bifurcates into posterior and dorsal branches. P, Q, T, U, In frac^Δ1 and frac^Δ2 homozygous mutants, axons make projections to aberrant muscle targets and are loosely bundled. J, N, R, V, Mmp2^W307* mutants display both defasciculation and targeting errors. A, B, Anterior is up; E–V, anterior is left and dorsal is up. Scale bars: A–D, 100 μm; E, F, 50 μm; G–V, 15 μm.

Figure 5.

frac is genetically downstream of Mmp2 in axon guidance. Two abdominal hemisegments of stage 17 embryos labeled with anti-FasII are shown with a schematic of the phenotype shown below. A, C, Wild-type embryos have tightly bundled axonal projections. B, D, In Mmp2^W307*^/+; frac^Δ1/+ embryos, the ISNb displays defasciculation defects. E, G, repo>Mmp2 embryos exhibit ISNb axons that are hyperfasciculated and fail to reach their targets. F, H, frac^Δ1, repo>Mmp2 embryos have axons that misproject and are defasciculated. I, J, In 24B>frac embryos, the ISNb displays extra projections and misrouted axons (arrows). Anterior is left and dorsal is up in all. Scale bar, 15 μm.

Figure 6.

Frac signals downstream through an LIMK-dependent BMP signaling mechanism. A–C, J–L, Two abdominal hemisegments of stage 17 embryos stained with anti-FasII to mark motor axon projections. A, D, frac^Δ1 mutant embryos display hypofasciculation and misprojection errors. B, E, In embryos expressing constitutively active Sax, the ISNb innervates its proper targets. C, F, Homozygous frac^Δ1 mutant embryos expressing elav>sax^act reach their targets with some defasciculation errors. Stage 16 wild-type (G), frac^Δ1 homozygous mutant (H), and 24B>frac (I) embryonic nerve cords labeled with pMad antibody. J, M, LIMK1^EY08757 homozygous mutants exhibit defasciculation errors. K, N, In embryos with neuronal RNAi knockdown of LIMK1 with concurrent neuronal misexpression of UAS–Dicer2, the ISNb displays targeting errors. L, O, The ISNb of LIMK1^EY08757; frac^Δ1 double mutant embryos is defasciculated and has ectopic projections. A–F, J–O, Anterior is left and dorsal is up. Scale bar: 15 μm. G–I, Anterior is up. Scale bars, 20 μm.

Figure 7.

Model of the interaction between Mmp2 and Frac during axon guidance. A, In wild type, both full-length Frac and Frac protein fragments bind an BMP molecule. Cleaved Frac fragments form a signaling complex with available BMP and signal axons to stay bundled. B, In Mmp2 LOF mutants, there is no protease to generate Frac fragments and therefore a signaling complex cannot form, resulting in increased defasciculation. C, In frac LOF mutants, there is no full-length or cleaved Frac present to bind the BMP molecule, preventing downstream signaling from occurring and resulting in increased defasciculation.