20-P027 The 5′–3′ exoribonuclease Pacman affects expression of JNK transcripts and has specific effects in Drosophila development

S312

MECHANISMS OF DEVELOPMENT

1 2 6 (2 0 0 9) S3 0 5–S 31 3

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causes similar pupal lethal phenotypes, suggesting that upper

Cell migration in vivo; determining the function of Ena

and lower threshold levels of PCM are important.

Philippa Tucker, Iwan Evans, Will Wood

doi:10.1016/j.mod.2009.06.859

University of Bath, Avon, United Kingdom Cell migration has been studied extensively in vitro, however, fewer studies address this subject in vivo. Our aim is to deter-

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mine in vivo, the role of the actin regulatory protein, Ena. To

The 50 –30 exoribonuclease Pacman affects expression of JNK

enable this we are studying Drosophila hemocytes in the devel-

transcripts and has specific effects in Drosophila development

oping embryo. Hemocytes are the primary immune cells of Dro-

Christopher I. Jones, Dominic P. Grima, Sarah F. Newbury

sophila and during development they follow stereotyped pathways to distribute throughout the embryo. Using the

Brighton and Sussex Medical School, Brighton, United Kingdom

Gal4UAS system to express GFP specifically in hemocytes allows

It is becoming increasingly clear that transcript degradation

us to visualize the migrating hemocytes using the confocal

via ribonucleases is an important step in the control of develop-

microscope. This system is also used to overexpress Ena or inter-

mental processes. Pacman (pcm) is the Drosophila homologue of

fere with its function. Here we show that Ena localizes to the

yeast XRN1 and is the only known cytoplasmic 50 –30 exoribonuc-

leading edge of the lamellipodia and tips of filopodia. Further-

lease in eukaryotes. To determine the effects of this exoribonuc-

more, Ena positively regulates filopodial and lamellipodial pro-

lease in development we have constructed a number of

trusions, as previously illustrated in fibroblasts in vitro.

mutants of Pacman and characterised the resulting phenotypes.

However, whereas Ena negatively regulates velocity of fibroblasts

A recently generated allele, where 3053 base pairs are deleted

in vitro it actually increases the velocity of hemocytes in vivo.

within the pcm coding region, results in 100% lethality at the

This key difference highlights the importance of studying gene

pupal stage, while smaller deletions result in less severe and via-

function in vivo.

ble phenotypes. As the viable mutant phenotypes are often similar to those

doi:10.1016/j.mod.2009.06.858

seen for mutants in the JNK signalling pathway, and Pacman has been shown to genetically interact with the phosphatise puckered, we reasoned that Pacman might target mRNAs in the

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JNK pathway. This pathway is involved in cell movement, for

Phenotypic consequences of pacman exoribonuclease misex-

example, during the developmental process dorsal closure and

pression in Drosophila development

during wound healing in adults. Thorax, wing and pupal develop-

Dominic P. Grima, Steven Hebbes, Maria V. Zabolotskaya,

ment are also frequently affected in Pacman strains. Taqman quantitative RT-PCR is being used to determine the

Sarah F. Newbury

extent of the effect of Pacman on the JNK pathway, and identify BSMS, University of Sussex, Falmer, Brighton, United Kingdom

other pathways in which it plays a role. Preliminary results show

Control of RNA stability has important implications for development. Drosophila developmental processes require the input of 0

the exoribonuclease pacman, which is the only known 5 to 3

0

cytoplasmic exoribonuclease in eukaryotes. These processes include dorsal closure, thorax closure, spermatogenesis and

the mRNA levels of some JNK pathway members are affected in Pacman mutants. This suggests Pacman may specifically target particular transcripts, suggesting a mechanism by which it can affect developmental processes. doi:10.1016/j.mod.2009.06.860

wound healing. Mutants in pacman show phenotypes which suggest an involvement with signalling pathways such as the JNK pathway, crucial to such epithelial closure pathways. Immunohistochemical staining with PCM antibodies reveals expression of

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pacman in the amnioserosa at dorsal closure, bolstering this

A fatemap of placodes in Xenopus laevis

hypothesis.

Mareike Pieper1, Gerhard Schlosser1,2

Our experiments to date reveal the first evidence of the

1

Brain Research Institute, University of Bremen, Bremen, Germany

involvement of an exoribonuclease in such developmental con-

2

National University of Ireland, Galway, Ireland

texts. This study examines the effects of mis-expressing PCM protein via transgenic UAS-PCM constructs carrying either wild-type

Many of the evolutionary novelties of the vertebrate head,

or nuclease dead forms of the PCM cDNA. Similar constructs con-

including specialised sense organs and neurons of the cranial

taining UAS-pcm-RNAi constructs were utilised to examine the

ganglia, arise from ectodermal placodes. These placodes become

effects of lowering PCM protein expression levels.

visible as local thickenings during neurulation. Several types of

By altering expression levels using both ubiquitous and tissue

placodes can be distinguished based on their fate and position:

specific GAL4 promoters, we show that PCM protein levels are

adenohypophyseal, otic, olfactory, trigeminal and profundal, lens,

important for morphogenesis of the developing embryo as well

a series of epibranchial and a series of lateral line placodes.

as affecting pupariation. Over-expression of the nuclease dead

Expression patterns of several genes including genes of the Six

form seems to lead to more severe phenotypes than the wild type

and Eya families suggest that all placodes have a common origin

construct, in line with preliminary data suggesting a dominant

from a region of the non-neural ectoderm situated around the

negative effect of such a construct. RNAi knockdown of PCM

anterior neural plate, termed the panplacodal primordium. In