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doi: 10.1242/10.1242/dev.00586

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Macrophage-mediated corpse engulfment is required for normal Drosophila CNS morphogenesis

Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue 68-230B, Cambridge, MA 02139, USA

View larger version (38K): [in a new window]   Fig. 1. Disruption of the Pvr locus through homologous recombination-mediated gene targeting. (A) A P-element containing 4.5 kb of Pvr genomic sequence, a mini-white gene and FRT sequences was randomly inserted by transposition onto the third chromosome. This DNA was circularized through expression of FLP recombinase and linearized through expression of the yeast restriction endonuclease I-SceI. This linear fragment recombined with the endogenous Pvr locus to produce two tandem partial copies of the Pvr gene. White boxes indicate exons that originate from the donor fragment. Black boxes indicate exons that originate from the endogenous locus. (B) PCR analysis of homologous recombinants using the primer pairs indicated in A. One primer of each pair anneals to DNA in the mini-white gene in the targeting construct, while the other primer of each pair anneals to genomic DNA from the endogenous locus. In the top panel, primers A and B were used to amplify DNA to the left of the mini-white gene. In the center panel, primers C and D were used to amplify DNA to the right of the mini-white gene. As expected, DNA from wild-type flies or flies with the donor P-element yield no PCR product in either case. DNA from homozygous Pvr knock-out embryos gives the expected size PCR products. In the bottom panel, control primers were used to amplify a fragment from the same genomic DNA used in the top and center panels. (C) Southern blot analysis of homologous recombinants. The locations and sizes of the fragments recognized by the probe are indicated in A. Red arrowhead, homologous recombinant-specific band; green arrowhead, endogenous Pvr locus band; blue arrowhead, pre-recombination donor-specific band. As PvrKO2, PvrKO4, and PvrKO10 are homozygous lethal early in development, DNA was obtained from adults heterozygous for each PvrKO allele and a wild-type copy of Pvr on a CyO chromosome. As expected, the endogenous Pvr locus band (green) is approximately twice as intense as the homologous recombinant-specific band (red), because the endogenous Pvr locus band is generated from both alleles.

View larger version (28K): [in a new window]   Fig. 2. Pvr alleles generated by EMS mutagenesis. (A) Schematic of the Pvr proteins noting the location of lesions predicted to severely truncate the Pvr protein. In Pvr9742, Q143 is converted to a stop codon. In Pvr5363, a 61-base deletion results in the deletion of amino acids 114-134 and a subsequent frameshift that results in three new amino acids and a stop codon. (B) Molecular lesions in Pvr alleles. Four alleles truncate the protein, four alleles contain missense mutations in the extracellular domain and three contain missense mutations in the kinase domain. (C) Western blots containing protein from the genotypes indicated probed with anti-Pvr antiserum. A single major protein species is detected in wild-type flies. This species cannot be detected in the Pvr mutants. Blots were reprobed with anti-Elav antiserum to confirm that similar or greater levels of protein were present in mutant lanes compared with wild-type controls.

View larger version (63K): [in a new window]   Fig. 3 Pvr is required for proper CNS morphogenesis. (A,B) Stage 16 (A) wild-type (Canton-S) and (B) Pvr5363 embryos stained with MAb BP102 to visualize CNS axons. ac, anterior commissure. pc, posterior commissure. lo, longitudinal tracts. Red line, CNS midline. In wild type, the commissures and longitudinal tracts form a rectangular axon scaffold in each segment (arrow), whereas in Pvr, the longitudinal tracts are closer together than in wild type, giving the axon scaffold a rounded appearance in each segment (arrow). (C,D) Stage 16 (C) wild type (Canton-S) and (D) Pvr5363 embryos stained with anti-Repo to visualize glia. In wild type, Repo-positive glia are arranged in a patterned array along the two longitudinal tracts, with a single row of Repo-positive glia between the longitudinal tracts in each segment (arrowhead). In Pvr mutants, Repo-positive glia accumulate at the midline (arrowheads), and there are fewer Repo-positive glia in the longitudinal tract region (arrow). The longitudinal tracts appear in outline in the photograph, which was taken using differential interference contrast (DIC) optics. (E,F) Longitudinal axon tracts of stage 17 embryos are stained with the anti-Fasciclin 2 monoclonal antibody 1D4. (E) Wild type. Three tightly bundled axon tracts can be seen on each side of the midline. (F) Pvr5363 mutants also form three axon tracts on either side of the midline. No ectopic axon crossing of the midline can be detected, although minor defasciculation of axon tracts can be seen in some segments (arrowhead).

View larger version (97K): [in a new window]   Fig. 4. Pvr protein is present on midline glia and hemocytes. (A-D) Wild-type stage 16 embryo stained with anti-Pvr (red) and anti-Peroxidasin (Pxn) (green). Yellow indicates overlap in the merged images (C,D). Pvr protein can be detected at the midline (arrowheads), in hemocytes (arrows) and at the surface of the embryo. (D) Higher magnification image of embryo depicted in A-C. The expression of Pvr in hemocytes is confirmed by co-expression with Peroxidasin in C and D. (E) Stage 17 embryo double-labeled with anti-Pvr (black) and BP102 (brown). Pvr protein is expressed on midline glia that surround the CNS commissures (arrowhead). (F) Higher magnification image of wild-type stage 16 embryo stained with anti-Pvr. CNS axon tracts are visualized using DIC optics. Pvr protein is expressed on midline glia (arrowheads) that surround the anterior and posterior CNS commissures. ac, anterior commissure. pc, posterior commissure. lo, longitudinal axons tracts. (G) PvrKO2 mutant embryo stained with anti-Pvr. Anti-Pvr staining cannot be detected in mutant animals.

View larger version (85K): [in a new window]   Fig. 5. Pvr acts in hemocytes and not midline glia for proper CNS patterning. (A,B) Stage 16 embryos stained with anti-wrapper antibody to visualize midline glia. Pvr5363 midline glia (B) are indistinguishable from wild type (A). (C,D) Stage 16 (C) Sim-Gal4;UAS-PvrDN and (D) Gcm-Gal4;UAS-PvrDN embryos stained with mAb BP102 to visualize CNS axons. Sim-Gal4;UAS-PvrDN embryos are indistinguishable from wild type, with commissures and longitudinal tracts forming a rectangular axon scaffold in each segment (arrow). Gcm-Gal4;UAS-PvrDN embryos resemble Pvr embryos, with the axon scaffold having a rounded appearance in each segment (arrow). (E,F) Stage 16 (E) Sim-Gal4;UAS-PvrDN and (F) Gcm-Gal4;UAS-PvrDN embryos stained with anti-Repo antibody to visualize glia. Sim-Gal4;UAS-PvrDN embryos have glia in normal positions, with only few glia located between commissures in each segment (arrowhead), while many segments of Gcm-Gal4;UAS-PvrDN embryos have clusters of glia near the midline (arrowheads). (G-I) Stage 16 (G) wild type (Canton-S), (H) PvrKO2 and (I) Gcm-Gal4;UAS-PvrDN embryos stained with anti-Croquemort to visualize hemocytes. In wild type, hemocytes are dispersed throughout the embryo, while in Pvr mutants and Gcm-Gal4;UAS-PvrDN embryos hemocytes are largely clustered near the dorsal and anterior regions of the embryo. Anterior is towards the left and dorsal is towards the top.

View larger version (146K): [in a new window]   Fig. 6. Macrophage function is required for proper CNS patterning. (A-D) Stage 16 (A) wild type, (B) PvrKO2,, (C) Srpneo45 and (D) Crq RNAi embryos stained with BP102 to visualize CNS axons. Srpneo45 and Crq RNAi embryos resemble Pvr embryos, with the axon scaffold having a rounded appearance in each segment (arrows). (E-H) Stage 16 (E) wild type, (F) PvrKO2,, (G) Srpneo45 and (H) Crq RNAi embryos stained with Repo to visualize glia. PvrKO2, Srpneo45 and Crq RNAi embryos all have disruptions in the pattern of Repo-positive glia. Srpneo45 mutant embryos have increased numbers of Repo-positive glia at the midline in each segment (arrowheads). In Crq RNAi embryos, there are also increased numbers of glia at the midline (arrowheads), while the glia associated with longitudinal tracts appear more dispersed than in wild type (arrow).