The exon junction complex is required for definition and excision of neighboring introns in Drosophila

Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon-exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization, and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity to either an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wild-type cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon desilencing in EJC-depleted Drosophila ovaries. Based on a transcriptome-wide analysis, we propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events

Pulses of Notch activation synchronise oscillating somite cells and entrain the zebrafish segmentation clock

Formation of somites, the rudiments of vertebrate body segments, is an oscillatory process governed by a gene-expression oscillator, the segmentation clock. This operates in each cell of the presomitic mesoderm (PSM), but the individual cells drift out of synchrony when Delta/Notch signalling fails, causing gross anatomical defects. We and others have suggested that this is because synchrony is maintained by pulses of Notch activation, delivered cyclically by each cell to its neighbours, that serve to adjust or reset the phase of the intracellular oscillator. This, however, has never been proved. Here, we provide direct experimental evidence, using zebrafish containing a heat-shock-driven transgene that lets us deliver artificial pulses of expression of the Notch ligand DeltaC. In DeltaC-defective embryos, in which endogenous Notch signalling fails, the artificial pulses restore synchrony, thereby rescuing somite formation. The spacing of segment boundaries produced by repetitive heat-shocking varies according to the time interval between one heat-shock and the next. The induced synchrony is manifest both morphologically and at the level of the oscillations of her1, a core component of the intracellular oscillator. Thus, entrainment of intracellular clocks by periodic activation of the Notch pathway is indeed the mechanism maintaining cell synchrony during somitogenesis

A genetic screen based on in vivo RNA imaging reveals centrosome-independent mechanisms for localizing gurken transcripts in Drosophila

We have screened chromosome arm 3L for ethyl methanesulfonate-induced mutations that disrupt localization of fluorescently labeled gurken (grk) messenger (m)RNA, whose transport along microtubules establishes both major body axes of the developing Drosophila oocyte. Rapid identification of causative mutations by single-nucleotide polymorphism recombinational mapping and whole-genomic sequencing allowed us to define nine complementation groups affecting grk mRNA localization and other aspects of oogenesis, including alleles of elg1, scaf6, quemao, nudE, Tsc2/gigas, rasp, and Chd5/Wrb, and several null alleles of the armitage Piwi-pathway gene. Analysis of a newly induced kinesin light chain allele shows that kinesin motor activity is required for both efficient grk mRNA localization and oocyte centrosome integrity. We also show that initiation of the dorsoanterior localization of grk mRNA precedes centrosome localization, suggesting that microtubule self-organization contributes to breaking axial symmetry to generate a unique dorsoventral axis

Imp/IGF2BP levels modulate individual neural stem cell growth and division through myc mRNA stability

The numerous neurons and glia that form the brain originate from tightly controlled growth and division of neural stem cells, regulated systemically by known extrinsic signals. However, the intrinsic mechanisms that control the characteristic proliferation rates of individual neural stem cells are unknown. Here, we show that the size and division rates of Drosophila neural stem cells (neuroblasts) are controlled by the highly conserved RNA binding protein Imp (IGF2BP), via one of its top binding targets in the brain, myc mRNA. We show that Imp stabilises myc mRNA leading to increased Myc protein levels, larger neuroblasts, and faster division rates. Declining Imp levels throughout development limit myc mRNA stability to restrain neuroblast growth and division, while heterogeneous Imp expression correlates with myc mRNA stability between individual neuroblasts in the brain. We propose that Imp-dependent regulation of myc mRNA stability fine-tunes individual neural stem cell proliferation rates

Greb1 is required for axial elongation and segmentation in vertebrate embryos

During vertebrate embryonic development, the formation of axial structures is driven by a population of stem-like cells that reside in a region of the tailbud called the chordoneural hinge (CNH). We have compared the CNH transcriptome with those of surrounding tissues and shown that the CNH and tailbud mesoderm are transcriptionally similar, and distinct from the presomitic mesoderm. Amongst CNH-enriched genes are several that are required for axial elongation, including Wnt3a, Cdx2, Brachyury/T and Fgf8 , and androgen/estrogen receptor nuclear signalling components such as Greb1 . We show that the pattern and duration of tailbud Greb1 expression is conserved in mouse, zebrafish, and chicken embryos, and that Greb1 is required for axial elongation and somitogenesis in zebrafish embryos. The axial truncation phenotype of Greb1 morphant embryos is explained by much reduced expression of No tail ( Ntl/Brachyury ) which is required for axial progenitor maintenance. Posterior segmentation defects in the morphants (including misexpression of genes such as mespb, myoD and papC ) appear to result, in part, from lost expression of the segmentation clock gene, her7

Greb1 is required for axial elongation and segmentation in vertebrate embryos

During vertebrate embryonic development, the formation of axial structures is driven by a population of stem-like cells that reside in a region of the tailbud called the chordoneural hinge (CNH). We have compared the mouse CNH transcriptome with those of surrounding tissues and shown that the CNH and tailbud mesoderm are transcriptionally similar, and distinct from the presomitic mesoderm. Amongst CNH-enriched genes are several that are required for axial elongation, including Wnt3a, Cdx2, Brachyury/T and Fgf8, and androgen/oestrogen receptor nuclear signalling components such as Greb1 We show that the pattern and duration of tailbud Greb1 expression is conserved in mouse, zebrafish and chicken embryos, and that Greb1 is required for axial elongation and somitogenesis in zebrafish embryos. The axial truncation phenotype of Greb1 morphant embryos can be explained by much reduced expression of No tail (Ntl/Brachyury), which is required for axial progenitor maintenance. Posterior segmentation defects in the morphants (including misexpression of genes such as mespb, myoD and papC) appear to result, in part, from lost expression of the segmentation clock gene, her7

Imp/IGF2BP levels modulate individual neural stem cell growth and division through myc mRNA stability

The numerous neurons and glia that form the brain originate from tightly controlled growth and division of neural stem cells, regulated systemically by important known stem cell-extrinsic signals. However, the cell-intrinsic mechanisms that control the distinctive proliferation rates of individual neural stem cells are unknown. Here, we show that the size and division rates of Drosophila neural stem cells (neuroblasts) are controlled by the highly conserved RNA binding protein Imp (IGF2BP), via one of its top binding targets in the brain, myc mRNA. We show that Imp stabilises myc mRNA leading to increased Myc protein levels, larger neuroblasts, and faster division rates. Declining Imp levels throughout development limit myc mRNA stability to restrain neuroblast growth and division, and heterogeneous Imp expression correlates with myc mRNA stability between individual neuroblasts in the brain. We propose that Imp-dependent regulation of myc mRNA stability fine-tunes individual neural stem cell proliferation rates

Cell-autonomous integrin control of Wnt and Notch signalling during somitogenesis

Integrins act at signalling crossroads, and their interactions with other signal transduction pathways are key to the regulation of normal and pathological cell cytoarchitecture and behaviour. Here, we describe a signalling cascade that acts during the formation of the defining segmental features of the vertebrate body – the somites – in which β1-integrin activity regulates epithelialisation by controlling downstream Wnt and Notch activity crucial for somite border formation. Using in vivo transcriptional inhibition in the developing chick embryo, we show that β1-integrin in the anterior presomitic mesoderm activates canonical Wnt signalling in a cell-autonomous, `outside-inside' manner. Signalling is mediated by integrin-linked kinase (ILK), leading to modulation of glycogen synthase kinase 3β (GSK3β) phosphorylation, and activates Notch signalling in the anterior presomitic mesoderm. The two signalling pathways then cooperate to promote somite formation via cMESO1/Mesp2. Our results show that β1-integrin can regulate cell shape and tissue morphogenesis indirectly, by regulation of downstream signalling cascades