Dynamically regulated transcription factors are encoded by highly unstable mRNAs in the larval brain.

The level of each RNA species depends on the balance between its rates of production and decay. Although previous studies have measured RNA decay across the genome in tissue culture and single-celled organisms, few experiments have been performed in intact complex tissues and organs. It is therefore unclear whether the determinants of RNA decay found in cultured cells are preserved in an intact tissue, and whether they differ between neighboring cell types and are regulated during development.

Neuronal upregulation of Prospero protein is driven by alternative mRNA polyadenylation and Syncrip-mediated mRNA stabilisation.

During and vertebrate brain development, the conserved transcription factor Prospero/Prox1 is an important regulator of the transition between proliferation and differentiation. Prospero level is low in neural stem cells and their immediate progeny, but is upregulated in larval neurons and it is unknown how this process is controlled. Here, we use single molecule fluorescent hybridisation to show that larval neurons selectively transcribe a long mRNA isoform containing a 15 kb 3' untranslated region, which is bound in the brain by the conserved RNA-binding protein Syncrip/hnRNPQ.

Syncrip/hnRNP Q is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation.

Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganization at new synapses, requiring posttranscriptional regulation of localized mRNA a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organizes actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is poorly understood.

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 , , and , and androgen/oestrogen receptor nuclear signalling components such as We show that the pattern and duration of tailbud expression is conserved in mouse, zebrafish and chicken embryos, and that is required for axial elongation and somitogenesis in zebrafish embryos.

Imp/IGF2BP levels modulate individual neural stem cell growth and division through 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 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, mRNA.

Imp and Syp RNA-binding proteins govern decommissioning of neural stem cells.

The termination of the proliferation of neural stem cells, also known as neuroblasts (NBs), requires a 'decommissioning' phase that is controlled in a lineage-specific manner. Most NBs, with the exception of those of the mushroom body (MB), are decommissioned by the ecdysone receptor and mediator complex, causing them to shrink during metamorphosis, followed by nuclear accumulation of Prospero and cell cycle exit. Here, we demonstrate that the levels of Imp and Syp RNA-binding proteins regulate NB decommissioning.

Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains.

RNA in situ hybridization is a powerful method to investigate post-transcriptional regulation, but analysis of intracellular mRNA distributions in thick, complex tissues like the brain poses significant challenges. Here, we describe the application of single-molecule fluorescent in situ hybridization (smFISH) to quantitate primary nascent transcription and post-transcriptional regulation in whole-mount Drosophila larval and adult brains. Combining immunofluorescence and smFISH probes for different regions of a single gene, i.

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.

Bicaudal-D1 regulates the intracellular sorting and signalling of neurotrophin receptors.

We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor-containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain-derived neurotrophic factor (BDNF)-activated TrkB and p75 neurotrophin receptor (p75(NTR)) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co-localisation of these neurotrophin receptors with retromer-associated sorting nexin 1. The resulting re-routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling-competent TrkB isoforms and p75(NTR) available for ligand binding on the neuronal surface.

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.

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.

Transcript processing and export kinetics are rate-limiting steps in expressing vertebrate segmentation clock genes.

Sequential production of body segments in vertebrate embryos is regulated by a molecular oscillator (the segmentation clock) that drives cyclic transcription of genes involved in positioning intersegmental boundaries. Mathematical modeling indicates that the period of the clock depends on the total delay kinetics of a negative feedback circuit, including those associated with the synthesis of transcripts encoding clock components [Lewis J (2003) Curr Biol 13(16):1398-1408]. Here, we measure expression delays for three transcripts [Lunatic fringe, Hes7/her1, and Notch-regulated-ankyrin-repeat-protein (Nrarp)], that cycle during segmentation in the zebrafish, chick, and mouse, and provide in vivo measurements of endogenous splicing and export kinetics.

Modifying transcript lengths of cycling mouse segmentation genes.

Regular production of somites, precursors of the axial skeleton and attached muscles is controlled by a molecular oscillator, the segmentation clock, which drives cyclic transcription of target genes in the unsegmented presomitic mesoderm (PSM). The clock is based on a negative feedback loop which generates pulses of transcription that oscillate with the same periodicity as somite formation. Mutants in several oscillating genes including the Notch pathway gene Lunatic fringe (Lfng) and the Notch target Hes7, result in defective somitogenesis and disorganised axial skeletons.

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.

Integrin-linked kinase is a functional Mn2+-dependent protein kinase that regulates glycogen synthase kinase-3β (GSK-3beta) phosphorylation.

Background: Integrin-linked kinase (ILK) is a highly evolutionarily conserved, multi-domain signaling protein that localizes to focal adhesions, myofilaments and centrosomes where it forms distinct multi-protein complexes to regulate cell adhesion, cell contraction, actin cytoskeletal organization and mitotic spindle assembly. Numerous studies have demonstrated that ILK can regulate the phosphorylation of various protein and peptide substrates in vitro, as well as the phosphorylation of potential substrates and various signaling pathways in cultured cell systems. Nevertheless, the ability of ILK to function as a protein kinase has been questioned because of its atypical kinase domain.

A'-form RNA helices are required for cytoplasmic mRNA transport in Drosophila.

Microtubule-based mRNA transport is widely used to restrict protein expression to specific regions in the cell and has important roles in defining cell polarity and axis determination as well as in neuronal function. However, the structural basis of recognition of cis-acting mRNA localization signals by motor complexes is poorly understood. We have used NMR spectroscopy to describe the first tertiary structure to our knowledge of an RNA element responsible for mRNA transport.

Identification and mapping of induced chromosomal deletions using sequence polymorphisms.

One of the many advantages of Drosophila melanogaster as a model organism is the relative ease with which gene deletions can be generated by imprecise excision of transposon insertions. Here, we describe a simple, fast, and efficient method of screening for single-gene excision events that is not biased by prior assumptions of the mutant phenotype. DNA sequence polymorphisms were used as co-dominant electrophoretic markers to identify candidate deletions in a single generation, and to delimit the breakpoints to within 0.

Differential axial requirements for lunatic fringe and Hes7 transcription during mouse somitogenesis.

Vertebrate segmentation is regulated by the "segmentation clock", which drives cyclic expression of several genes in the caudal presomitic mesoderm (PSM). One such gene is Lunatic fringe (Lfng), which encodes a modifier of Notch signalling, and which is also expressed in a stripe at the cranial end of the PSM, adjacent to the newly forming somite border. We have investigated the functional requirements for these modes of Lfng expression during somitogenesis by generating mice in which Lfng is expressed in the cranial stripe but strongly reduced in the caudal PSM, and find that requirements for Lfng activity alter during axial growth.

The Groucho/TLE/Grg family of transcriptional co-repressors.

The Drosophila Groucho (Gro) protein was the founding member of the family of transcriptional co-repressor proteins that now includes the transducin-like enhancer of split (TLE) and Grorelated gene (Grg) proteins in vertebrates. Gro family proteins do not bind DNA directly, but are recruited by a diverse profile of transcription factors, including members of the Hes, Runx, Nkx, LEF1/Tcf, Pax, Six and c-Myc families. The primary structure of Gro proteins includes five identifiable regions, of which the most highly conserved are the amino-terminal glutamine-rich Q domain and the carboxy-terminal WD-repeat domain.

Differential in vivo requirements for oligomerization during Groucho-mediated repression.

The Groucho (Gro)/transducin-like enhancer of split family of transcriptional corepressors are implicated in many signalling pathways that are important in development and disease, including those mediated by Notch, Wnt and Hedgehog. Here, we describe a genetic screen in Drosophila that yielded 50 new gro alleles, including the first protein-null allele, and has two mutations in the conserved Q oligomerization domain that have been proposed to have an essential role in corepressor activity. One of these latter mutations, encoding an amino-terminal protein truncation that lacks part of the Q domain, abolishes oligomerization in vitro and renders the protein unstable in vivo.

Molecular recognition of transcriptional repressor motifs by the WD domain of the Groucho/TLE corepressor.

The Groucho (Gro)/TLE/Grg family of corepressors operates in many signaling pathways (including Notch and Wnt). Gro/TLE proteins recognize a wide range of transcriptional repressors by binding to divergent short peptide sequences, including a C-terminal WRPW/Y motif (Hairy/Hes/Runx) and internal eh1 motifs (FxIxxIL; Engrailed/Goosecoid/Pax/Nkx). Here, we identify several missense mutations in Drosophila Gro, which demonstrate peptide binding to the central pore of the WD (WD40) beta propeller domain in vitro and in vivo.

Upregulation of chicken p15INK4b at senescence and in the developing brain.

In mammalian cells, products of the INK4a-ARF locus play major roles in senescence and tumour suppression in different contexts, whereas the adjacent INK4b gene is more generally associated with transforming growth factor beta (TGF-beta)-mediated growth arrest. As the chicken genome does not encode an equivalent of INK4a, we asked whether INK4b and/or ARF contribute to replicative senescence in chicken cells. In chicken embryo fibroblasts (CEFs), INK4b levels increase substantially at senescence and the gene is transcriptionally silenced in two spontaneously immortalised chicken cell lines.

The epsilon-subunit of mitochondrial ATP synthase is required for normal spindle orientation during the Drosophila embryonic divisions.

We describe the maternal-effect and zygotic phenotypes of null mutations in the Drosophila gene for the epsilon-subunit of mitochondrial ATP synthase, stunted (sun). Loss of zygotic sun expression leads to a dramatic delay in the growth rate of first instar larvae and ultimately death. Embryos lacking maternally supplied sun (sun embryos) have a sixfold reduction in ATP synthase activity.

Inscuteable mRNA localization is dynein-dependent and regulates apicobasal polarity and spindle length in Drosophila neuroblasts.

Drosophila neuroblasts undergo asymmetric divisions along the apicobasal axis to produce two daughter cells of unequal size and different developmental fate. Inscuteable (Insc) protein functions as part of an apically localized complex to coordinate orientation of the mitotic spindle and basal sorting of cell fate determinants. insc mRNA transcripts also localize apically in neuroblasts, yet the mechanism underpinning this process and its developmental significance are unknown.