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.

A central challenge in embryonic development is to understand how growth and pattern are coordinated to direct emerging new territories during morphogenesis. Here, we report on a signaling cascade that links cell proliferation and fate, promoting formation of a distinct progenitor domain within the developing chick hypothalamus. We show that the downregulation of Shh in floor plate-like cells in the forebrain governs their progression to a distinctive, proliferating hypothalamic progenitor domain. Shh downregulation occurs via a local BMP-Tbx2 pathway, Tbx2 acting to repress Shh expression. We show in vivo and in vitro that forced maintenance of Shh in hypothalamic progenitors prevents their normal morphogenesis, leading to maintenance of the Shh receptor, ptc, and preventing progression to an Emx2(+)-proliferative progenitor state. Our data identify a molecular pathway for the downregulation of Shh via a BMP-Tbx2 pathway and provide a mechanism for expansion of a discrete progenitor domain within the developing forebrain.

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SUMMARYThe infundibulum links the nervous and endocrine systems, serving as a crucial integrating centre for body homeostasis. Here we describe that the chick infundibulum derives from two subsets of anterior ventral midline cells. One set remains at the ventral midline and forms the posterior-ventral infundibulum. A second set migrates laterally, forming a collar around the midline. We show that collar cells are composed of Fgf3 + SOX3 + proliferating progenitors, the induction of which is SHH dependent, but the maintenance of which requires FGF signalling. Collar cells proliferate late into embryogenesis, can generate neurospheres that passage extensively, and differentiate to distinct fates, including hypothalamic neuronal fates and Fgf10 + anterior-dorsal infundibular cells. Together, our study shows that a subset of anterior floor plate-like cells gives rise to Fgf3 + SOX3 + progenitor cells, demonstrates a dual origin of infundibular cells and reveals a crucial role for FGF signalling in governing extended infundibular growth.