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.

Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. Our results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates.

The formation of the dorsoventral (DV) boundary is central to establishing the body plan in embryonic development. Although there is some information about how limbs are positioned along the DV axis and how DV skin color pattern is determined, the way in which mammary glands are positioned is unknown. Here we focus on Bmp4 and Tbx3, a gene associated with ulnar-mammary syndrome, and compare their expression along the DV axis in relation to mammary gland initiation in mouse embryos. Tbx3 is expressed in the mammary gland-forming region with Tbx15, a gene involved in a DV coat color being expressed more dorsally and Bmp4 being expressed more ventrally. When Tbx3 was overexpressed, formation of mammary gland epithelium was extended along the DV axis. In contrast, overexpression of Bmp4 inhibited both Tbx3 and Tbx15 expression. In addition, when BMP signaling was inhibited by NOGGIN, Lef1 expression was lost. Thus, we propose that mutual interactions between Bmp4 and Tbx3 determine the presumptive DV boundary and formation of mammary glands in early mouse embryogenesis. 1,19-Dioctadecyl-3,3,39,39-tetramethyl indocarbocyanine perchloride labeling experiments showed that cells associated with mammary glands originate more dorsally and then move ventrally. This finding, together with previous findings, suggests that the same DV boundary may not only position limbs and determine coat color but also position mammary glands. Furthermore, Bmp signaling appears to be a fundamental feature of DV patterning.dorsoventral patterning ͉ ulnar-mammary syndrome

Members of the spalt family of zinc finger-containing proteins have been implicated in development and disease. However, very little is known about the molecular function of spalt proteins. We have used biochemical approaches to characterize functional domains of two chick spalt homologs, csal1 and csal3. We show that csal1 and csal3 proteins repress transcription and that they can interact with each other. Furthermore, we found that truncated chick spalt proteins, similar to the truncated spalt protein expressed in the human congenital disorder Townes-Brocks syndrome, affect the nuclear localization of full-length spalt. Our findings have implications for the understanding of Townes-Brocks syndrome and the role of spalt genes in normal development. We propose that truncated spalt can exert a dominant negative effect and is able to interfere with the correct function of full-length protein, by causing its displacement from the nucleus. This could affect the transcriptional repressor activity of spalt and DNA binding. Spalt protein truncations could also affect the function of other spalt family members in various tissues.This work focuses on the functional analysis of two chick homologs of the spalt family, csal1 and csal3.