RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo.

During early vertebrate development Fibroblast Growth Factor (FGF) signalling is required for multiple activities including specification of mesodermal, neural and heart tissue, as well as gastrulation movements and regulation of differentiation and pattern onset in the extending body axis. A current challenge is to understand how FGF signalling generates such diverse outcomes. A key FGF downstream pathway is the Ras-MAPK/Erk1/2 cascade, which culminates in the phosphorylation of target proteins, such as the Ets family of transcription factors. To begin to assess specificity downstream of FGF in the chick embryo we have characterised the patterns of Fgfr1-4 expression and Erk1/2 activation, as well as expression of the Erk1/2 specific phosphatase, Mkp3 and of three Ets factor genes (Erm, Pea3 and Er81) from early blastula to the 10 somite stage. We identify new sites of Fgfr expression and show that nearly all regions of Erk1/2 activity are within Fgfr expression domains and require FGF signalling. Differences in intensity, duration, distribution and sub-cellular localisation of activated Erk1/2 are observed in distinct cell populations within the embryo and during wound healing. With few exceptions, a tight correspondence between Erk1/2 activation and Mkp3 expression is found, while specific combinations of Ets factors are associated with distinct regions of Erk1/2 activation. These findings provide a comprehensive spatial and temporal map of FGF/Erk1/2 activity during early chick development and identify region and tissue specific differences in expression of Fgfrs as well as Erk1/2 phosphorylation and transcriptional targets which help to define response specificity.

Generation of the spinal cord relies on proliferation of undifferentiated cells located in a caudal stem zone. Although fibroblast growth factor (FGF) signaling is required to maintain this cell group, we do not know how it controls cell behavior in this context. Here we characterize an overlooked expression domain of the Notch ligand, Delta1, in the stem zone and demonstrate that this constitutes a proliferative cell group in which Notch signaling is active. We show that FGF signaling is required for expression of the proneural gene cash4 in the stem zone, which in turn induces Delta1. We further demonstrate that Notch signaling is required for cell proliferation within the stem zone; however, it does not regulate cell movement out of this region, nor is loss of Notch signaling sufficient to drive neuronal differentiation within this tissue. These data identify a novel role for the Notch pathway during vertebrate neurogenesis in which signaling between high Delta1-expressing cells maintains the neural precursor pool that generates the spinal cord. Our findings also suggest a mechanism for the establishment of the cell selection process, lateral inhibition: Mutual inhibition between Delta/Notch-expressing stem zone cells switches to single Delta1-presenting neurons as FGF activity declines in the newly formed neuroepithelium.[Keywords: Notch signaling; FGF signaling; proneural genes; spinal cord; stem cells; lateral inhibition] Supplemental material is available at http://www.genesdev.org.

1. G protein-regulated inward rectifier K¤ (GIRK) channels were over-expressed in dissociated rat superior cervical sympathetic (SCG) neurones by co-transfecting green fluorescent protein (GFP)-, GIRK1-and GIRK2-expressing plasmids using the biolistic technique. Membrane currents were subsequently recorded with whole-cell patch electrodes. 2. Co-transfected cells had larger Ba¥-sensitive inwardly rectifying currents and 13 mV more negative resting potentials (in 3 mÒ [K¤]o) than non-transfected cells, or cells transfected with GIRK1 or GIRK2 alone. 3. Carbachol (CCh, 1-30 ìÒ) increased the inwardly rectifying current in 70% of GIRK1+ GIRK2-transfected cells by 261 ± 53 % (n = 6, CCh 30 ìÒ) at −120 mV, but had no effect in non-transfected cells or in cells transfected with GIRK1 or GIRK2 alone. Pertussis toxin prevented the effect of carbachol but had no effect on basal currents. 4. The effect of CCh was antagonized by 6 nÒ tripitramine but not by 100 nÒ pirenzepine, consistent with activation of endogenous Mµ muscarinic acetylcholine receptors. 5. In contrast, inhibition of the voltage-activated Ca¥ current by CCh was antagonized by 100 nÒ pirenzepine but not by 6 nÒ tripitramine, indicating that it was mediated by MÚ muscarinic acetylcholine receptors. 6. We conclude that endogenous Mµ and MÚ muscarinic receptors selectively couple to GIRK currents and Ca¥ currents respectively, with negligible cross-talk.8938