embryonic stem cells and their transitions during differentiation are regulated by miRNAs, we analysed the miRNA expression pro- file of ES cells as they differentiate toward an extra-embryonic ecto- derm fate (trophoblast) following acute downregulation of the stem cell master regulator Oct4. The expression levels of approximately 5% of the identified miRNAs changed markedly upon Oct4 down- regulation. We are currently performing experiments designed to interfere with the function of these developmentally-regulated miRNAs, and investigating the roles that specific miRNAs play in proliferation and differentiation of ES cells. doi:10.1016/j.mod.2009.06.021 01-P021 Transcriptional complexity at the murine Hoxa5 locus Lucie Jeannotte 1 , Yan Coulombe 1 , Margot Lemieux 1 , Julie Moreau 1 , Sbastien Tabaris 1 , Franois Guillou 1 , Milan Joksimovic 2 , Christopher K. Tuggle 2 , Jose Aubin 1 1 Centre de recherche en cancrologie de l’Universit Laval, L’Htel-Dieu de Qubec, Qubec, QC, Canada 2 Department of Animal Science, Iowa State University, Ames, IO, United States Hox gene functions are intimately linked to their correct devel- opmental expression. We are studying the Hoxa5 gene, which plays a crucial role in axial patterning and organogenesis. In the mouse embryo, five transcripts of 1.8, 5, 9 and 11 kb in length are associated with the Hoxa5 locus, the 1.8 kb transcript being the most abundant and corresponding to the two known exons of the gene. Molecular characterization of the different tran- scripts encompassing Hoxa5 sequences has revealed that distal transcription start sites located in the Hoxa6-Hoxa7 intergenic region combined with alternative splicing are responsible for the production of the larger forms. Comparative insitu hybridiza- tion showed that some structures, like the cervico-upper thoracic prevertebrae and the lung, exclusively express the 1.8 kb mRNA. This expression pattern correlates with that of the HOXA5 pro- tein, supporting the notion that the 1.8 kb transcript is the func- tional form of the Hoxa5 gene. The larger transcripts are expressed later during embryogenesis and in more posterior structures than the 1.8 kb transcript. Their expression profile is similar to that of the Hoxa7 gene suggesting that they may share some DNA regulatory elements with the Hoxa7 promoter. Even though, the larger transcripts include Hoxa5 coding sequences, there is no evidence that they produce the Hoxa5 protein, raising the possibility that they may act as noncoding RNAs. Our results will be discussed in the context of the intricate organization of the Hoxa5-Hoxa7 region of the HoxA cluster. (Supported by CIHR) doi:10.1016/j.mod.2009.06.022 01-P022 Functional analysis of the neuronal Hu RNA-binding proteins Peter McCarthy , Kate Dredge, Kirk Jensen The University of Adelaide, Adelaide, South Australia, Australia Of the four Hu genes found in most vertebrates (HuA, HuB, HuC & HuD), all except HuA exhibit mRNA and protein expression that is essentially restricted to post-mitotic neurons of the devel- oping and adult nervous systems. Spatial and temporal examina- tion of individual ‘‘neuronal Hu’’ (nHu; nHu = HuB, HuC and HuD) proteins in brain tissue suggests nHu proteins may play a func- tional role during neuronal differentiation; as RNA binding pro- teins, the nHu proteins may participate in gene regulatory events that are essential for acquisition of the neuronal phenotype. We have identified a number of candidate mRNA targets of the nHu proteins. Our data suggests that the majority of these mRNAs interact with nHu proteins through sequences present in their 3 untranslated regions (UTRs). From this 3 UTR target sub- set, several mRNAs were selected for further examination based on reported roles for their encoded proteins during axonogenesis, including mRNAs for cytoskeleton-modifying proteins cofilin, vasodilator stimulated phosphoprotein (VASP) and the Rho GTPase Cdc42. We have placed the 3UTR sequences from each of our selected mRNAs downstream of a Luciferase coding sequence to test for an effect of nHu proteins on target mRNA expression. In co-trans- fection assays with HuC, we observe a small but significant decrease in target 3UTR-reporter protein expression. Subsequent measurement of 3UTR-reporter mRNA levels suggest this effect is potentially the result of changes in 3UTR polyadenylation that have an effect on reporter translatability. Regulation of mRNA polyadenylation may be a novel mechanism by which nHu pro- teins control mRNA translation during neuronal development. doi:10.1016/j.mod.2009.06.023 01-P023 A noncoding function of oskar RNA in Drosophila oogenesis Ronald Gstir , Helena Jambor, Anne Ephrussi European Molecular Biology Laboratory, Heidelberg, Germany In Drosophila, the embryonic body axes are predefined by local- ized maternal RNAs and proteins in the oocyte. Localization of oskar mRNA and its translational repression during transport to the oocyte posterior pole are critical for correct formation of the abdomen and germ cells in the embryo. Remarkably, oskar has an additional role as a noncoding RNA during oogenesis, prior to the onset of Oskar protein expression. In oskar RNA null flies, oogenesis is affected during the early stages and arrests at stage 6–7, although the developing oocyte is properly determined and polarized. Loss of oskar mRNA also causes karyosome fragmenta- tion and impairs Staufen protein accumulation in the oocyte. We wish to understand the molecular function of oskar as a non-coding RNA and how it directly impacts on oogenesis. To this end, we are characterizing the oskar RNA null phenotype in greater detail and have found that genetically modulating the protein levels of the translational repressor Bruno can restore karyosome integrity. By transgenic analysis, we have shown that 166 nucleotides of the oskar 3 untranslated region (UTR) targeted to the oocyte are sufficient to rescue the oogenesis arrest of the oskar RNA null mutant. To gain insight into the processes regu- S57 MECHANISMS OF DEVELOPMENT 126 (2009) S51 – S59