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First published online 26 March 2008
doi: 10.1242/dev.020685

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Smt3 is required for Drosophila melanogaster metamorphosis

Ana Talamillo^1,*, Jonatan Sánchez^1,*, Rafael Cantera^2,3, Coralia Pérez¹, David Martín⁴, Eva Caminero⁵ and Rosa Barrio^1,

¹ Functional Genomics Unit, CIC bioGUNE, Technology Park, Building 801-A, 48160 DERIO, Bizkaia, Spain.
² Zoology Department, Stockholm University, 10691 Stockholm, Sweden.
³ Instituto de Investigaciones Biológicas Clemente Estable, Av. Italia, 3318 Montevideo, Uruguay.
⁴ Institut de Biologia Molecular de Barcelona, CSIC, J. Girona 18-26, 08034 Barcelona, Spain.
⁵ Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, 28049 Madrid, Spain.

View larger version (51K): [in this window] [in a new window]   Fig. 1. Smt3 role on development and metamorphosis. (A) Cartoon of the ring gland, a neuroendocrine complex located above the brain, composed of: the PG cells that produce the ecdysone hormone (blue); the corpus allatum cells that produce the juvenile hormone (purple); and the corpora cardiaca (red). The PG is innervated by neurosecretory cells depicted in red (only one hemisphere has been represented). (B,C) Ring gland staining showing nuclei (purple) and Smt3 expression (green). (B',C') Green channels showing Smt3 expression only are shown in black and white. Boxed regions are magnified in B'' and C''. Smt3 levels are strongly reduced in smt3i nuclei (C',C'') compared with wild type (B',B''), although some residual protein can be observed in smt3i PG cells (arrows). (D-F) smt3i larvae do not pupariate but continue growing as larvae, becoming approximately double the weight at 18-21 days AEL (F). (G,H) The morphology and the number of teeth (arrows) in mouth hooks indicate that smt3i larvae reached the third instar and stayed at that stage throughout the rest of their prolonged larval life.

View larger version (27K): [in this window] [in a new window]   Fig. 2. smt3i larvae show low ecdysteroid levels. (A) Graph of the levels of ecdysteroids (E and 20E) in larvae of different genotypes at different times AEL, expressed in pg per mg of larvae. The peak of ecdysteroids that in WT, phm-Gal4 and UAS-smt3i larvae is thought to induce pupariation is not found in smt3i larvae. (B) smt3i larvae fed with 20E can pupariate. Pupae proceeded to head eversion, but in no instance gave rise to adults.

View larger version (80K): [in this window] [in a new window]   Fig. 3. The PGs of smt3i larvae do not show massive degeneration. (A-D) Confocal single plane micrographs showing DAPI staining of nuclei (purple) and Phalloidin (green) to show cell contour. The nuclei of smt3i cells (B-D) show a large DAPI-negative space in the centre. Cells are bigger than in WT (A) and some `giant cells' appear in each PG (arrows). With time, smt3i PGs show lower number of cells than do WT (C,D). (E-G) Confocal pictures showing nuclei (purple) and the activated initiator caspase Nc (green) that indicates whether the cell has entered into apoptosis. At 5 to 6 days AEL, the PGs do not show massive cell death and the levels of Nc are comparable to WT (E versus F). Only sporadic apoptotic cells are observed in smt3i larvae, which could correspond to the `giant cells' (arrow in G). (H) Electron micrograph showing four apoptotic bodies (AB), in a large extracellular space probably representing the remnants of an apoptotic cell, surrounded by non-apoptotic cells (labelled a, b and c) in a 5-day-old smt3i ring gland. Scale bar: 2 µm. He, hemocoel; BL, basal lamina.

View larger version (120K): [in this window] [in a new window]   Fig. 4. smt3i PG cells show changes in steroidogenic enzymes and transcription factors. (A-O) Single plane confocal micrographs showing expression of the indicated hydroxylase enzymes (Phm, Dib or Sad) or the indicated transcription factors (Woc, Mld or βFtz-f1) in WT, smt3i or lwr mutant PG cells. Nuclei are labelled with DAPI and shown in purple; the indicated proteins are shown in green. (A'-O') Single green channels for each panel, showing expression of the indicated proteins, are presented in black and white. White arrows denote differences in the nuclear accumulation of the referred factors between WT and knockdown phenotypes, and white arrowheads denote ectopic cytoplasmic accumulation of certain factors in knockdown or mutant backgrounds. (A,A',D,D') Phm, accumulated in the ER, does not show changes in expression levels or localisation in smt3i larvae compared with WT, whereas Dib mitochondrial staining is reduced (B,B',E,E'). Sad nuclear accumulation, but not mitochondrial staining, is reduced in knockdown larvae (C,C',F,F', arrows). (G,J,M) Woc nuclear localisation does not diminish in smt3i (J,J') or lwr mutant larvae (M,M') compared with WT (G,G'). In lwr mutants, Woc accumulates in the cytoplasm (arrowhead). (H,K,N) Mld accumulates in the nucleus in WT cells (H,H', arrow), and is highly reduced in smt3i nuclei (K,K', arrow), although cytoplasmic accumulations of the protein can be observed (arrowheads). This is even more noticeable in lwr mutant larvae (N,N', arrowheads). (I,L,O) βFtz-f1 appears to be evenly localised in the nucleus (arrow) and the cytoplasm in WT cells (I,I'), whereas in smt3i (L,L') and lwr mutant larvae (O,O') it is reduced in the cytoplasm and absent in the nuclei (arrows). All images are shown at the same magnification.

View larger version (67K): [in this window] [in a new window]   Fig. 5. The structure of the cell membrane is compromised in smt3i PG cells. (A-D) Transmission electron microscopy images of PG cells from WT (A,C) and smt3i (B,D) larvae. (A,B) Intracellular channels (IC, arrows) are severely reduced in number and size in smt3i larvae (B) compared with WT (A), although no differences were observed in number, size or morphology of the mitochondria (Mi). BL, basal lamina. (C,D) The nuclear lamina is thickened in smt3i (D) compared with WT (C), but the nuclear pores appear not to be affected (arrows). Nu, nucleus; Cy, cytoplasm. Scale bars: 500 nm. (E,F) Single confocal micrographs showing PG cell nuclei stained with DAPI (purple) and anti-HRP antibodies (green). (E',F') Single green channels for HRP staining shown in black and white.

View larger version (103K): [in this window] [in a new window]   Fig. 6. The lipid content of smt3i PG cells is reduced. (A,B) Confocal micrographs showing nuclei marked with DAPI (blue) and lipid droplets stained with Oil Red O (red) in WT (A) and knockdown (B) larvae. Single red channels are shown in black and white (A',B'), and boxed regions are magnified (A",B"). Most smt3i PG cells contain very few droplets (arrow in B"), except for the `giant cells', which accumulate large drops (arrow in B'). (C,D) Filipin staining shows a reduction in sterol droplets (white dots) in smt3i PG cells (D) compared with the number of droplets in WT (C). Arrows in C indicate cells containing several lipid droplets in WT cells, whereas arrows in D indicate remnant lipid droplets in some cells of the smt3i PG. (E) Graph of the average number of Oil Red O droplets per cell present in a single confocal plane in WT versus smt3i PG cells, reflecting a one-third reduction of droplets.

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