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Full Abstracts – GAMETOGENESIS AND ORGANOGENESIS
175
155
Cell-type-specific translational control of
cycB
in the
Drosophila
male germline.
Catherine C. Baker, Byung Soo Gim, Margaret T. Fuller. Dept
Developmental Biol, Stanford Univ Sch Medicine, Stanford, CA.
In the
Drosophila
male germline, the majority of the mRNAs required for post-meiotic spermatid differentiation are transcribed in spermatocytes, the male
germ cells undergoing meiotic G2 prophase. This spermatocyte transcription program and a 20-fold increase in cell volume require that G2 prophase be
extended for a total of 3.5 days before the meiotic divisions occur. We have found that the delay of meiotic division is directed in part by cell type-specific
regulation of Cyclin B (CycB), a member of the core cell cycle machinery. The mRNA for CycB is expressed throughout spermatocyte development, but
CycB protein does not appear until just before meiotic division. We have found that this delay in appearance of CycB is due to translational repression by
Rbp4, an RNA-binding protein. When
rbp4
is knocked down by RNAi, CycB protein appears in early spermatocytes where it is normally absent. Rbp4 binds
to a 35-nucleotide (nt) conserved sequence within the short (130nt) spermatocyte 3’UTR of
cycB
, and that same conserved sequence is also required for
translational repression of a CycB-eYFP reporter in early spermatocytes. With the help of BioGRID and FlyAtlas, we identified a potential co-factor:
CG9975 (nicknamed Fest). Fest and Rbp4 co-immunoprecipitate from S2 cells, and both proteins are expressed in early spermatocytes onwards.
Surprisingly, Fest does not function with Rbp4 to repress translation of
cycB
, but acts instead in opposition: CycB does not accumulate when
fest
is knocked
down in spermatocytes by RNAi. To test whether (a) Fest antagonizes Rbp4 in late spermatocytes to allow translation of
cycB
, or (b) Rbp4 antagonizes
translation-promoting activity of Fest until late spermatocytes, we expressed the Rbp4-resistant CycB-eYFP reporter (Δ35nt) in a
fest
RNAi background.
The CycB-eYFP(Δ35nt) reporter was derepressed in early spermatocytes in the wild-type control, but expression was not detected in
fest
RNAi
spermatocytes. This suggests that Fest is directly required for
cycB
translation, with Rbp4 likely repressing its activity in all but the most mature
spermatocytes.
156
Barriers to Male Transmission of Mitochondrial DNA in Sperm Development.
Steven A. DeLuca. Dev Biol, UCSF, San Francisco, CA.
The mitochondrial genome (mtDNA) is maternally inherited in most animals, and studies in a few animals have proposed that paternal mtDNA elimination
is coupled to zygote formation. We investigated the molecular mechanisms responsible for eliminating paternal mtDNA in Drosophila, and unexpectedly
found that paternal inheritance is prevented prior to zygote formation. Using a genetic tool developed in our lab (Xu et al. Science, 2008), we isolated
mtDNA mutations that allowed us to follow paternal mtDNA through a cross. We discovered that fertilized embryos lacked paternal mtDNA, and mtDNA
was also absent in mature sperm. We then documented two distinct processes that eliminate mtDNA from developing sperm. We visualized the abrupt
disappearance of mtDNA nucleoids during the last 100 microns of sperm tail elongation, and identified a mitochondrial nuclease, Endonuclease G, that is
required for this disappearance. In Endonuclease G mutants, persisting mtDNA nucleoids were collected and eliminated by a second process that trims and
shapes spermatid tails during sperm individualization. Our results document the primary mechanisms enforcing a unique inheritance strategy in which the
father restricts the transmission of his own mtDNA.