doi: 10.1242/10.1242/dev.00454
fear of intimacy encodes a novel transmembrane protein required for gonad morphogenesis in Drosophila
Mark Van Doren1,2,*,
,
Wendy R. Mathews1,*,
Monique Samuels2,
Lisa A. Moore2,
,,
Heather Tarczy Broihier2,
and
Ruth Lehmann2
1 Department of Biology, 305 Mudd Hall, Johns Hopkins University, 3400 N.
Charles Street, Baltimore, MD 21218, USA
2 Howard Hughes Medical Institute, Developmental Genetics Program, Skirball
Institute at NYU School of Medicine, 540 First Avenue, New York, NY 10016,
USA
Present address: Incyte Genomics, 3160 Porter Drive, Palo Alto, CA 94304,
USA
Present address: Department of Genetics, Washington University School of
Medicine, 4566 Scott Avenue, St Louis, MO 63110, USA
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Fig. 6. Subcellular localization of HA-FOI. (A) Deconvolution microscopy image of a
Schneider S2 cell expressing HA-FOI immunolabeled with an anti-HA antibody
(red) and stained with DAPI to reveal the nucleus (DNA, blue). Note that the
FOI protein is localized to the plasma membrane. (B) Deconvolution microscopy
image of a stage 14 embryonic gonad expressing HA-FOI in the germ cells,
immunostained for a germ cell marker (anti-VASA, green) and HA-FOI (red).
HA-FOI is predominantly localized to the germ cell surface. (C) Confocal image
of a stage 16 embryonic gonad expressing HA-FOI in the mesoderm (expression
driven by twist 24B gal4), including gonadal mesoderm, immunostained to label
the gonadal mesoderm (anti-ZFH-1, red) and HA-FOI (green). Note that HA-FOI is
observed surrounding each germ cell (asterisks), although the germ cells are
not expressing HA-FOI in this experiment. HA-FOI partially co-localizes with
DLG at the surface of gonadal mesoderm cells (inset) in a stage 16 embryo
immunostained to label DLG (red), HA-FOI (green, expression driven by 24B
Gal4) and germ cells (anti-vasa, blue). (D) Confocal image of a stage 15
embryo expressing HA-FOI in the trachea, immunostained to reveal the tracheal
lumen (Mab 2A12, green) and HA-FOI (red). Lateral trunk branches are shown.
HA-FOI appears to localize to the cell periphery and also to extensions
between fusion tip cells during branch fusion (inset). In all panels shown,
the HA-tag is in the N-terminal region of FOI.
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Fig. 4. FOI protein and selected FICL family members. Protein domains are as
indicated in the key. Arrowheads above FOI show the position of nonsense
mutations found in the three EMS-induced foi mutants. Percentages
reflect the percent identical amino acids within the designated domains when
compared to FOI or CATSUP as indicated.
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Fig. 1. foi mutants are blocked in gonad coalescence. (A,B) Stage 14
embryos immunostained with an anti-VASA antibody (brown) that labels the germ
cells. Dorsal view, anterior left. (A) Wild-type embryo. Note that the germ
cells have properly coalesced into the embryonic gonad. (B) foi
mutant embryo. The germ cells have aligned on either side of the embryo, but
have failed to coalesce into the gonad. (C) Confocal microscopy image showing
the gonad region of a stage 14 foi mutant embryo double immunolabeled
to reveal the germ cells (anti-VASA, red) and the gonadal mesoderm (anti-EYA,
green). Note that although gonad coalescence is blocked, the germ cells have
migrated to, and are correctly associating with, the gonadal mesoderm.
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Fig. 2. Gonadal mesoderm phenotype of foi and E-cadherin/shg.
Stage 14-15 embryos labeled by in situ hybridization to reveal expression of
the 412 retrotransposon (brown), a gonadal mesoderm marker. (A) Wild type. The
gonadal mesoderm has coalesced with the germ cells to form the embryonic
gonad. (B,C) foi16.33 mutant embryos showing examples of
the severe (B) or moderate (C) gonad coalescence defects. Note that in B, the
gonadal mesoderm cells fail to coalesce and, instead, extend into the
neighboring mesoderm. In C, the gonadal mesoderm partially coalesces with the
germ cells, but the gonad does not attain the compact, spherical appearance
observed in wild type. (D,E) Embryos from osk mutant mothers
(foi20.71 oskCE4/osk301) aged at
18°C that lack germ cells but otherwise exhibit normal embryonic
patterning. Embryo in E is also zygotically mutant for foi
(foi20.71 oskCE4/foi16.33). Note
that the gonadal mesoderm coalesces normally in the absence of germ cells (D),
but that the foi mutant gonad phenotype is still readily apparent
under these conditions (E). (F) Embryo zygotically mutant for
shgIH (E-cadherin). Note that the gonadal
mesoderm fails to fully coalesce into an embryonic gonad.
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Fig. 3. foi mutants show defects in tracheal branch fusion. (A) Schematic
of the developing tracheal system highlighting lateral trunk (LT) fusion
during stages 14 and 15. Drawing courtesy of Mark Krasnow and is modified from
Manning and Krasnow (Manning and Krasnow,
1993 ). GB, ganglionic branch. (B-D) Confocal images of stage 16
embryos (breathless Gal4 x UAS-mCD8-GFP) immunostained to label the
tracheal cells (anti-GFP, red) and the tracheal lumen (Mab 2A12, green). (B)
Wild-type embryo. (C,D) foi20.71 mutant embryos. Short
arrows indicate segments where tracheal branch fusion has occurred and long
arrows indicate segments where fusion has failed to occur. Note that the
ganglionic branches still extend ventrally (down) from segments with failed
fusion. Inset in D highlights protrusions between fusion tip cells in a
segment where fusion failed to occur. (E) Confocal image of a stage 15
foi20.71 mutant embryo containing the
escargotG66B enhancer trap, immunostained to reveal
enhancer trap expression (anti-ß-gal, red) and the tracheal lumen (Mab
2A12, green). Asterisks indicate fusion tip cells expressing the esg
enhancer trap. Note that the fusion tip cells from the segment that has failed
to fuse still express the enhancer trap. Short arrows indicate segments where
tracheal branch fusion has occurred and long arrows indicate segments where
fusion has failed to occur.
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Fig. 5. foi RNA expression pattern. Embryos labeled by in situ
hybridization revealing the foi RNA expression pattern (purple). (A)
Stage 3 embryo. The maternal foi RNA has almost completely
disappeared except for in the pole cells (posterior, right). (B) Stage 6
embryo. foi is expressed generally but is enriched in the
invaginating mesoderm. (C) Stage 9 embryo. Note the high levels of
foi RNA in the anterior and posterior midgut primordia. (D) Stage 14
embryo. foi is broadly expressed, but only low levels are found in
the epidermis.
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Fig. 7. Tissue-specific rescue of the foi mutant phenotypes. In all cases,
embryos are mutant for the endogenous foi gene
(foi20.71) and rescue experiments were conducted as
described in the Materials and Methods. Gal4 indicates the tissue expressing
the Gal4 transcriptional activator (meso, mesoderm; gc, germ cells). UAS
indicates whether a UAS-foi expression transgene was present and
which one. (A) Rescue of the foi gonad phenotype. Expression of
foi in the mesoderm fully rescues the foi mutant gonad
defect, while expression of foi in the germ cells shows no rescue.
(B) Rescue of the foi tracheal phenotype. Expression of foi
in either the trachea or mesoderm rescues the tracheal fusion defect. (C,D)
Stage 15 gonads immunostained to reveal the germ cells (anti-VASA, red) and
the gonadal mesoderm (anti-EYA, green). (C) foi mutant showing
defective gonad coalescence. (D) foi mutant rescued by expression of
HA-FOI (C-terminal tag) in the mesoderm. Coalescence is normal.
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© The Company of Biologists Ltd 2003
