Molecular characterization of Pegarn: a Drosophila homolog of UNC-51 kinase

Abstract

We have isolated and characterized the gene encoding a Drosophila melanogaster homolog of Caenorhabditis elegans UNC-51 (uncoordinated movement-51): Pegarn. Developmental Northern blot shows the Pegarn gene is expressed at all stages of development. The protein is detected throughout the Drosophila third instar larval central nervous system (CNS) in axons projecting out from the ventral ganglion and in the optic anlagen of the optic lobe. Heterozygous Pegarn mutant embryos show defects in larval axonal neuronal patterning, but survive to adulthood. Homozygous mutants have an even more deformed pattern of neuronal development and do not survive through the larval stages. The data from this research suggest the critical roles of Pegarn in CNS and PNS axonal formation in Drosophila melanogaster and indicates its similar role in other multicellular species.

Fig. 1

(a) Amino acid alignment of C. elegans UNC-51, human ULK1, mouse ULK1, and Pegarn by the ClustalW program. Identical residues are shaded. (b) Genomic organization and relative positions of the mutant EP lines. Pegarn kinase contains 9 introns (green boxes). The sizes of introns a to i are approximately, 6,500, 9,000, 160, 160, 60, 50, 347, 60, and 60 bp respectively. Exons are represented by pink boxes. The exons sizes from I to X are 788, 174, 87, 126, 72, 162, 282, 1080 141, and 236 bp respectively. Also shown in this figure are the relative location of P element insertions of the EP lines, EP(3)3348 and EP(3)3193

Fig. 2

Developmental Northern Blot of Pegarn. (a) Total RNA from 4 h, 12 h, 1 day, 2 day, 4 day, 6 day, 8 day and adult: embryos and flies were transferred to the Nitrocellulose membrane and probed with Pegarn cDNA. (b) Total RNA of adult wild type fly head and body. (c) Total RNA from mutants EP(3)3348, EP(3)3193 and wild type flies were used. EP(3)3348 heterozygote flies were over a balancer (TM6B). The arrow indicates 3.5 kb transcripts. The lower panels of each blot are the controls for loading of RP49

Fig. 3

Western blotting of head and body of wild type fly, heads of EP(3)3348 and EP(3)3193. Protein was extracted from the head and body of wild type flies, heads of EP(3)3348 and EP(3)3193 and Western blot analysis was performed with anti-Pegarn purified antibody. The lists of the numbers on the side of each figure represent molecular weight marker. (a) Lane 1, 2 and 3 are wild type heads, wild type bodies, and heterozygous EP(3)3348 heads respectively. Antibody to tubulin for the loading control is shown at the bottom of the Fig. 3. (b) Lane 1 and 2 are wild type heads and EP(3)3193 homozygous heads respectively. Antibody to tubulin was used for the detection of protein loading and shown in the same blot

Fig. 4

Expression pattern of Pegarn protein in wild type animals by immunofluorescence staining. (a) Adult wild type fly heads with purified Pegarn antibody, (b) pre-immune serum, (c) larval CNS axon (in the brain hemisphere): purified Pegarn antibody, (d) pre-immune on same tissue, (e–j) expression pattern in the axons projecting out from the ventral ganglion co-staining with monoclonal antibody 22C10 (red), Pegarn antibody (green) and merge (yellow) vs. (k) pre-immune. The images of Fig. 4e–g and h–j are taken from the same ventral ganglion but from different focal planes. The thorax and (l) the abdomen (n) of adult fly bodies with purified Pegarn antibody vs. (m, o) pre-immune sera around the same area

Fig. 5

Immunofluorescence of third instar larvae brain hemisphere. (a–c) Wild type, (d–f) heterozygous mutant, (g–i) homozygous mutant. Each row represents Pegarn staining (green), 22C10 staining (red) and merge (yellow). The expression patterns were seen in the neuropils of the optic lobe anlagen with reduced expression level in homozygote mutants. EP(3)3348 heterozygote flies were over a balancer (TM6B)

Fig. 6

Immunofluorescence of the wild type 12-h embryos. (a–c) Double staining of 22C10 and Pegarn. The 22C10 (red) staining in (a), Pegarn (green) in (b) and merge (yellow) in (c). (d–f) Double staining of 12-h embryos with monoclonal antibody BP102 (red) and Pegarn (green) and merge (yellow). (g) Pre-immune staining

Fig. 7

Immunofluorescence of 12-h mutant embryos (a–i and k–l) and j (wild type embryo). (a–f) Heterozygote mutant embryos co-staining of 22C10 (red) with Pegarn (green), and merge (yellow). All EP(3)3348 flies were heterozygote over a balancer (TM6B). (g–i) Homozygote mutant embryos co-staining of 22C10 (red) and Pegarn (green) and merge (yellow). The secondary antibody used for Pegarn were Cy5 but further processed by Adobe photoshop and changed the color from blue to green channel. (j–l) The images of BP102 staining in wild type, heterozygote and homozygote Pegarn mutant embryos respectively. EP(3)3348 heterozygote flies were over a balancer (TM6B)