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First published online June 28, 2004
doi: 10.1242/10.1242/dev.01229

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Mechanosensory-defective, male-sterile unc mutants identify a novel basal body protein required for ciliogenesis in Drosophila

James D. Baker, Sreedevi Adhikarakunnathu^* and Maurice J. Kernan

Department of Neurobiology and Behavior and Center for Developmental Genetics, Stony Brook University, Stony Brook, NY 11794-5230, USA

View larger version (183K): [in a new window]   Fig. 1. Spermatogenesis defects in unc mutants. (A,B) Cysts of elongating spermatids in wild-type (A) and unc mutant (B) testis, stained for nuclei (red) and ß-tubulin (blue). Nuclei are clustered at the head of wild-type cysts but are dispersed in unc mutants. (C) Nuclei in cysts of an unc mutant testis, stained for DNA (red) and -tubulin (blue). Two stages of nuclear condensation are present. Earlier, less-condensed nuclei (left side of panel) are still associated with the -tubulin-containing centriole adjuncts as in wild type, but mutant nuclei (arrows) and adjuncts (arrowheads) frequently detach at later stages. Mutant nuclei do not condense further than the stage seen in cross-section on the right. (D-F) Ultrathin sections of wild-type (D) and mutant (E,F) testes. Wild-type spermatids have a canonical 9+2 axoneme, associated with an electron-dense mitochondrial derivative. Most mutant axonemes are partially or completely disrupted, and lose their tight association with mitochondrial derivatives. Scale bars: in A-C, 5 µm; in D,F, 0.2 µm.

View larger version (167K): [in a new window]   Fig. 2. Defective sensory cilia in unc mutants. (A-F) Sensory neurons in the antenna (A-D) and the femoral chordotonal organ (E,F), labeled by neuronal expression of mCD8-GFP. Arrows indicate ciliary outer dendritic segments, and arrowheads to the tips of the inner dendritic segments. In wild type, cilia extend from sensory processes of olfactory neurons on the antenna (A, enlarged in B) and femoral chordotonal neurons (E). In unc mutants (C,D,F) neurons and inner sensory processes are still present, but the cilia are missing or truncated. (G-L) ultrathin sections of individual sensory units (scolopidia) in chordotonal organs. (G) Transverse sections of wild-type scolopidia, showing profiles of two cilia (ci) enclosed in each scolopale (sc). (H,I) unc mutant femoral (H) or antennal (I) scolopidia with missing or disrupted cilia (box and inset). Sections through some basal bodies (I) also showed breaks (inset). (J) Schematic of a scolopidium, relating numbered section planes to the sections seen in G-I. (K,L) Longitudinal sections of wild-type and mutant ciliary bases. Basal bodies appear as paired electron-dense structures (arrowhead); the distal basal body was often less clearly defined in mutant cilia. Scale bars: 20 µm in A,C; 5 µm in E; 1 µm in G; 0.2 µm in K.

View larger version (26K): [in a new window]   Fig. 3. unc encodes a large coiled-coil protein. (A) Complementation and physical maps of the unc region at the base of the X chromosome. Flies heterozygous for the overlapping deficiencies Df(1)S54 and Df(1)B57 show an unc phenotype. The left breakpoint of Df(1)S54 and breakpoints associated with three x-ray-induced unc alleles are indicated. (B) The unc transcription unit, with promoter fusion, rescuing transgene and GFP fusion constructs below. The open reading frame of a cDNA clone (LP08350) was extended by 5'-RACE to a 5' UTR with in-frame stop codons. A transgene including 2 kb of upstream DNA fused to the balance of the cDNA restored normal sound-evoked responses (C) to unc mutants. The same upstream DNA fragment was fused to EGFP to report transcription from the unc promoter. To examine protein localization, DNA encoding EGFP was appended to the unc-coding sequence. (D) Predicted unc proteins from D. melanogaster (D.m.), D. pseudoobscura (D.p.) and the mosquito Anopheles gambiae (A.g.). Each protein includes several short coiled-coil segments, separated by proline residues or proline-rich regions, and a Lissencephaly 1 homology (LisH) motif (green box). The positions of stop codons in five EMS-induced unc alleles are indicated in the melanogaster protein. (E) Alignment of the LisH motifs from the proteins in D and from the human ODF1 protein (H.s.).

View larger version (57K): [in a new window]   Fig. 4. unc-driven GFP expression in testes and sensory neurons. (A) Apical end of adult testis with GFP fluorescence (green) and autofluorescence from spermatid mitochondria (blue). The unc promoter region drives visible GFP expression in spermatocytes, but not in the early gonial or stem cells at the apical tip (arrow). (B-E) GFP expression in peripheral sensory neurons of the embryo (B,C) and pupal wing margin (D,E), visualized with anti-GFP (green) and the neuronal marker Futsch (MAb22C10, red). unc is also expressed in the campaniform sensilla of the wing blade, but these cells appear red because of strong expression of Futsch. Scale bars: 40 µm.

View larger version (76K): [in a new window]   Fig. 5. UNC-GFP localization in sensory neurons. (A,B) A singly innervated macrochaete bristle showing a focus of Unc-GFP labeling (arrow) near the end of the sensory process. The neuronal process and the ciliary outer segment are labeled with anti-HRP (red); autofluoresence from the cuticle outlines the bristle and socket in green. (C) Clustered spots of Unc-GFP (green) label the multiply innervated olfactory sensilla on the third antennal segment. Nuclei are labeled with propidium iodide (red). The boxed region is enlarged in the inset, without the nuclear labeling. (D) Two embryonic lateral chordotonal clusters. Anti-22C10 (red) labels the neuronal and inner dendritic segments, but not the ciliary outer segments. Each neuron shows two labeled spots (green, arrows), with the more proximal at the end of the inner segment. Scale bars: in A, 1 µm for A,B; in C, 20 µm for C, 4 µm for D.

View larger version (73K): [in a new window]   Fig. 6. Unc-GFP localization in spermatogenesis. Confocal images of spermatids (A-C) and spermatocytes or gonial cells (D-G) expressing UNC-GFP (green). Anti-ß-tubulin (blue) labels axonemal and cytoplasmic microtubules in all panels, and propidium iodide (red) stains DNA in B-D. (A) Low-magnification image of the of the testis tip, showing UNC-GFP in elongated spermatids at the apical end of the flagella. (B) Round spermatids immediately following meiosis II show a single GFP-labeled centriole associated with each nucleus. (C) UNC-GFP is associated with the centriole adjunct (arrowhead) in elongating spermatids, but is not present in mature spermatids with fully condensed nuclei (open arrowhead). (D) Primary spermatocytes in meiosis I, showing paired centrioles at each pole of the meiotic spindle. (E,F) Co-expression of UNC-GFP and centrosomin (red) in gonial cells (arrow). (G) Colocalization of UNC-GFP and centrosomin (red) in dividing spermatocytes. Scale bars: 10 µm in A; 5 µm in B-G.

View larger version (84K): [in a new window]   Fig. 7. Unc-GFP is first localized in G2 spermatocytes. Merged phase-contrast and fluorescence image of spermatocytes from larval testes expressing UNC-GFP. (A) Timeline of spermatocyte development. (B-G) Polar, apolar and mature spermatocytes in a wild-type testis. UNC-GFP is first localized in polar spermatocytes to four paired dots (B,C; enlarged in inset); the labeled structures are larger in apolar spermatocytes (D,E) and migrate to the cell periphery in mature cells (F,G). (H,I) Arrested gonial cells in bgcn mutant testis: no localized Unc-GFP is detectable. (J,K) Arrested primary spermatocytes in an sa mutant: paired UNC-GFP labeled structures are smaller than those in wild-type cells at a comparable stage. Scale bar: 20 µm.

View larger version (136K): [in a new window]   Fig. 8. Altered -tubulin localization in unc mutant spermatocytes. (A) Unc-GFP (green) and anti--tubulin (blue) labeling cysts at multiple stages of development in a wild-type testis. DNA is labeled with propidium iodide (red). Boxes, enlarged below, include cells entering meiosis I (1) and exiting meiosis II (2). (B,C) -Tubulin labeling in wild-type (B)and unc mutant (C) spermatocytes. In mutants, -tubulin staining is reduced to small dots, each probably equivalent to a pair of centrioles in wild type. Scale bars: 10 µm.

View larger version (65K): [in a new window]   Fig. 9. Reduced centrioles in unc mutant spermatocytes. Transverse and longitudinal sections of centrioles in (A) wild-type (y w) and (B) mutant (y w unc) spermatocytes show similar ultrastructure with microtubule triplets, but both the proximal and distal sections of mutant centrioles are shorter. (C) Lengths of the proximal and distal parts of longitudinally sectioned centrioles. Asterisks indicate the centrioles shown in A and B.

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