Ken Howard

Absence of effects of Sir2 overexpression on lifespan in C. elegans and Drosophila

Overexpression of sirtuins (NAD+-dependent protein deacetylases) has been reported to increase lifespan in budding yeast (Saccharomyces cerevisiae), Caenorhabditis elegans and Drosophila melanogaster. Studies of the effects of genes on ageing are vulnerable to confounding effects of genetic background. Here we re-examined the reported effects of sirtuin overexpression on ageing and found that standardization of genetic background and the use of appropriate controls abolished the apparent effects in both C. elegans and Drosophila. In C. elegans, outcrossing of a line with high-level sir-2.1 overexpression abrogated the longevity increase, but did not abrogate sir-2.1 overexpression. Instead, longevity co-segregated with a second-site mutation affecting sensory neurons. Outcrossing of a line with low-copy-number sir-2.1 overexpression also abrogated longevity. A Drosophila strain with ubiquitous overexpression of dSir2 using the UAS-GAL4 system was long-lived relative to wild-type controls, as previously reported, but was not long-lived relative to the appropriate transgenic controls, and nor was a new line with stronger overexpression of dSir2. These findings underscore the importance of controlling for genetic background and for the mutagenic effects of transgene insertions in studies of genetic effects on lifespan. The life-extending effect of dietary restriction on ageing in Drosophila has also been reported to be dSir2 dependent. We found that dietary restriction increased fly lifespan independently of dSir2. Our findings do not rule out a role for sirtuins in determination of metazoan lifespan, but they do cast doubt on the robustness of the previously reported effects of sirtuins on lifespan in C. elegans and Drosophila.

Transcription pattern of the Drosophila segmentation gene hairy

Segmentation of the Drosophila embryo requires expression of the pair-rule genes, mutations of which cause reiterated deletions in alternate segments along the antero-posterior body axis. We find that transcripts of one such gene, hairy, accumulate in eight distinct regions of the early embryo. This pattern of expression is compared with that of another pair-rule gene, fushi tarazu, and its dependence on maternally expressed genes is described.

Regulatory interactions between the segmentation genes fushi tarazu, hairy, and engrailed in the Drosophila blastoderm

The genetic loci fushi tarazu (ftz), hairy (h), and engrailed (en) must be expressed during embryogenesis for the correct segmental organization of the Drosophila embryo to be established. Transcripts from these genes accumulate in periodic patterns at the blastoderm stage of development. We demonstrate that generation of the normal pattern of ftz RNA requires the h+ function whereas formation of the h pattern does not require the ftz gene. In addition, we show that the en pattern is altered in both h and ftz mutant embryos. The nature of these changes and the cuticular phenotype of h mutant larvae suggest that both h and ftz phenotypes are a consequence of incorrect compartmentalization and that the effect of h is mediated via ftz and other genes.

Fly and mammalian lipid phosphate phosphatase isoforms differ in activity both in vitro and in vivo

Wunen (Wun), a homologue of a lipid phosphate phosphatase (LPP), has a crucial function in the migration and survival of primordial germ cells (PGCs) during Drosophila embryogenesis. Past work has indicated that the LPP isoforms may show functional redundancy in certain systems, and that they have broad‐range lipid phosphatase activities in vitro, with little apparent specificity between them. We show here that there are marked differences in biochemical activity between fly Wun and mammalian LPPs, with Wun having a narrower activity range than has been reported for the mammalian LPPs. Furthermore, although it is active on a range of substrates in vitro, mouse Lpp1 has no activity on an endogenous Drosophila germ‐cell‐specific factor in vivo. Conversely, human LPP3 is active, resulting in aberrant migration and PGC death. These results show an absolute difference in bioactivity among LPP isoforms for the first time in a model organism and may point towards an underlying signalling system that is conserved between flies and humans.

Lipid phosphate phosphatases dimerise, but this interaction is not required for in vivo activity

BackgroundLipid phosphate phosphatases (LPPs) are integral membrane proteins believed to dephosphorylate bioactive lipid messengers, so modifying or attenuating their activities. Wunen, a Drosophila LPP homologue, has been shown to play a pivotal role in primordial germ cell (PGC) migration and survival during embryogenesis. It has been hypothesised that LPPs may form oligomeric complexes, and may even function as hexamers. We were interested in exploring this possibility, to confirm whether LPPs can oligomerise, and if they do, whether oligomerisation is required for either in vitro or in vivo activity.ResultsWe present evidence that Wunen dimerises, that these associations require the last thirty-five C-terminal amino-acids and depend upon the presence of an intact catalytic site. Expression of a truncated, monomeric form of Wunen in Drosophila embryos results in perturbation of germ cell migration and germ cell loss, as observed for full-length Wunen. We also observed that murine LPP-1 and human LPP-3 can also form associations, but do not form interactions with Wunen or each other. Furthermore, Wunen does not form dimers with its closely related counterpart Wunen-2. Finally we discovered that addition of a trimeric myc tag to the C-terminus of Wunen does not prevent dimerisation or in vitro activity, but does prevent activity in vivo.ConclusionLPPs do form complexes, but these do not seem to be specifically required for activity either in vitro or in vivo. Since neither dimerisation nor the C-terminus seem to be involved in substrate recognition, they may instead confer structural or functional stability through dimerisation. The results indicate that the associations we see are highly specific and occur only between monomers of the same protein.

Organogenesis: Drosophila goes gonadal

In many animals, germ cells migrate to the gonad to assemble into a functional organ. Recent work in Drosophila has built a picture of the gene activities that specify the gonad and allow it to attract germ cells, and has led to the identification of a gene, columbus, that may encode the attractive factor.

Developmental biology. Attractive genetics.

During development, the precursors for eggs and sperm — the germ cells — migrate to the gonads to mature. But what attracts them there? Results from a fruitfly screen indicate that one component in this pathway is a gene calledcolumbus, which encodes HMG-CoA reductase. In humans, this enzyme is involved in cholesterol biosynthesis, raising the intriguing possibility that the attractant may be a lipid.

The view from the top.

San Diego is facing significant challenges to its future development. How do the some of the regions leading lights think it will cope? Ken Howard finds out.