Screening for gene function in chicken embryo using RNAi and electroporation

Pekařík, Vladimír; Bourikas, Dimitris; Miglino, Nicola; Joset, Pascal; Preiswerk, Stephan; Stoeckli, Esther T.

doi:10.1038/nbt770

Cited by 190 publications

(135 citation statements)

References 19 publications

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“…Before implantation, beads were rinsed in PBS and soaked in a solution of 0.1 g͞ l FGF8 mouse recombinant protein (R&D Systems) overnight at 4°C. dsRNA (25) were synthesized from cDNA encoding Fgf8 (26); single strands of either sense or antisense Fgf8 RNA were used for control series. Delivery of RNA interference (RNAi) molecules was achieved by in ovo electroporation into the 5-6ss chicken embryo.…”

Section: Methodsmentioning

confidence: 99%

Reciprocal relationships between Fgf8 and neural crest cells in facial and forebrain development

Creuzet

Schuler

Couly

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

148

128

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Fgf8 exerts a strong effect on the mesenchymal cells of neural crest (NC) origin that are fated to form the facial skeleton. Surgical extirpation of facial skeletogenic NC domain (including mid-diencephalon down through rhombomere 2), which does not express Hox genes, results in the failure of facial skeleton development and inhibition of the closure of the forebrain neural tube, while Fgf8 expression in the telencephalon and in the branchial arch (BA) ectoderm is abolished. We demonstrate here that (i) exogenous FGF8 is able to rescue facial skeleton development by promoting the proliferation of NC cells from a single rhombomere, r3, which in normal development contributes only marginally to mesenchyme of BA1, and (ii) expression of Fgf8 in forebrain and in BA ectoderm is subjected to signal(s) arising from NC cells, thus showing that the development of cephalic NC-derived structures depends on FGF8 signaling, which is itself triggered by the NC cells.cephalic neural crest ͉ facial skeleton ͉ forebrain ͉ regeneration ͉ quail-chick chimeras I n vertebrates, the skeleton and connective components of the face are derived from the cephalic neural crest (NC), which can be divided into two domains. The first, a rostral domain, in which no Hox genes are expressed, extends from the presumptive level of the epiphysis down through the second rhombomere (r2); it yields the cartilages and membrane bones of the face (Fig. 1 A). It is referred to here as the facial skeletogenic NC (FSNC). The second, posterior domain (from r4 through r8) generates part of the hyoid cartilages and does not form any membrane bone. In this posterior domain of the NC, Hox genes of the four first paralogous groups are expressed both in neural tube and NC (1-3). A few NC cells (NCC) from r3 contribute to both domains. However, this contribution to branchial arches (BAs) is very small because most r3-derived NCC are undergoing apoptosis (4, 5).When the entire Hox-negative domain of the NC is removed, no facial structures develop, meaning that Hox-expressing NCC do not substitute for the Hox-negative ones (6, 7). In contrast, any fragment of the Hox-negative crest, grafted in the anterior cephalic region following the ablation of the FSNC, can regenerate a normal face (6). Thus, the Hox-negative crest behaves as an equivalence group showing that, at the early stages, the crest cells themselves do not possess the information to construct the specific bones and cartilages that constitute the facial skeleton. The ventrolateral endoderm of the foregut is able to provide the NCC with the information necessary for patterning the facial skeleton and also the hyoid cartilage (6,8). Later in development of the facial structures, the ectoderm of the facial process also participates in the final patterning of the beak (9, 10).The investigations reported here were prompted by the observation that removal of the FSNC resulted in a dramatic decrease of Fgf8 expression in the forebrain anlage, as well as in the BA ectoderm ( Fig. 1 B-E). This was followed by the t...

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Section: Methodsmentioning

confidence: 99%

Reciprocal relationships between Fgf8 and neural crest cells in facial and forebrain development

Creuzet

Schuler

Couly

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

148

128

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“…Alternatively, we used a combination of dsRNA (300 ng/μl) and a plasmid encoding GFP (25 ng/μl) in PBS with 0.02% Fast Green (AppliChem). For electroporation, we used the same settings as described previously (Pekarik et al, 2003). Embryos were sacrificed at the desired HH stage and their spinal cords were analyzed as open-book preparations as described (Perrin and Stoeckli, 2000).…”

Section: In Ovo Rnai and Open-book Preparationmentioning

confidence: 99%

Calsyntenin 1-mediated trafficking of axon guidance receptors regulates the switch in axonal responsiveness at a choice point

Alther¹,

Domanitskaya²,

Stoeckli³

2016

Journal of Cell Science

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Axon guidance at choice points depends on the precise regulation of guidance receptors on the growth cone surface. Upon arrival at the intermediate target or choice point, a switch from attraction to repulsion is required for the axon to move on. Dorsal commissural (dI1) axons crossing the ventral midline of the spinal cord in the floor plate represent a convenient model for the analysis of the molecular mechanism underlying the switch in axonal behavior. We identified in chick a role for calsyntenin 1 in the regulation of vesicular trafficking of guidance receptors in dI1 axons at choice points. In cooperation with RabGDI, calsyntenin 1 shuttles Rab11-positive vesicles containing Robo1 to the growth cone surface in a precisely regulated manner. By contrast, calsyntenin 1-mediated trafficking of frizzled 3, a guidance receptor in the Wnt pathway, is independent of RabGDI. Thus, tightly regulated insertion of guidance receptors, which is required for midline crossing and the subsequent turn into the longitudinal axis, is achieved by specific trafficking.

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“…In this study, we examined the role of 2-O-sulfation in the morphogenesis of the chicken limb. Electroporation of RNA duplexes (RNAi technique) is effective at suppressing the expression of target genes in chick embryos (35). Disruption of the HS2ST gene by small inhibitory RNA (siRNA) reduced 2-O-sulfation levels and led to the abnormal development of limb buds, which showed less expression of both Fgf-8 and Fgf-10 accompanied by the up-regulation of phosphorylation of both ERK and Akt.…”

mentioning

confidence: 99%

Essential Role of Heparan Sulfate 2-O-Sulfotransferase in Chick Limb Bud Patterning and Development

Kobayashi

Habuchi

Tamura

et al. 2007

Journal of Biological Chemistry

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Although growth factors and morphogens are required for limb bud development and patterning, it is unclear how their signaling and distribution are regulated. Heparan sulfate proteoglycans, which are ubiquitous on the cell surface and in the extracellular matrix including basement membranes, are important regulators of such growth factor signaling and distribution (9 -11). For example, chicken syndecan-3 is expressed throughout the distal mesenchymal cells and plays a role in limb bud outgrowth by controlling FGF signaling between the AER and mesoderm (12). Heparan sulfate proteoglycans play such roles by interacting with these growth factors (13-15); however, it is unclear what role HS chains play in these processes, although HS chains are known to interact with a variety of heparin-binding growth factors (HBGFs) such as FGF.In FGF signaling, the ternary complex composed of HS chains, FGF family molecules, and FGF receptors (FGFRs) is formed on the cell surface (16 -18). Specificities of interactions between heparan sulfate proteoglycan and ligand molecules are thought to reside in the fine structures of HS chains with specific sequences consisting of highly sulfated monosaccharides (N-, 2-O-, and/or 6-O-sulfated) (9,19,20). In fact, the different O-sulfation patterns of HS chains are involved in interactions with FGF family molecules (21-23). These fine structures are generated by the following modification reactions with specific enzymes after the backbone chain of HS is synthesized by the addition of alternating D-glucuronic acid and N-acetyl-D-glucosamine (GlcNAc) residues from the respective UDP sugars by EXT family proteins (24): GlcNAc N-deacetylation and N-sulfation by N-deacetylase/N-sulfotransferase, C-5 epimerization of D-glucuronic acid residues to iduronic acid by C-5 epimerase, and O-sulfations at various places that take place first at C-2 of IdoA and D-glucuronic acid by heparan sulfate 2-O-sulfotransferase (HS2ST), subsequently at C-6 of GlcNAc and N-sulfate-*

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Screening for gene function in chicken embryo using RNAi and electroporation

Cited by 190 publications

References 19 publications

Reciprocal relationships between Fgf8 and neural crest cells in facial and forebrain development

Reciprocal relationships between Fgf8 and neural crest cells in facial and forebrain development

Calsyntenin 1-mediated trafficking of axon guidance receptors regulates the switch in axonal responsiveness at a choice point

Essential Role of Heparan Sulfate 2-O-Sulfotransferase in Chick Limb Bud Patterning and Development

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