Zhang S et al. (2013), A functional genome-wide in vivo screen identif...

XB-ART-47770

PLoS One 2013 Jan 01;811:e79469. doi: 10.1371/journal.pone.0079469.

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A functional genome-wide in vivo screen identifies new regulators of signalling pathways during early Xenopus embryogenesis.

Zhang S , Li J , Lea R , Amaya E , Dorey K .

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Embryonic development requires exquisite regulation of several essential processes, such as patterning of tissues and organs, cell fate decisions, and morphogenesis. Intriguingly, these diverse processes are controlled by only a handful of signalling pathways, and mis-regulation in one or more of these pathways may result in a variety of congenital defects and diseases. Consequently, investigating how these signalling pathways are regulated at the molecular level is essential to understanding the mechanisms underlying vertebrate embryogenesis, as well as developing treatments for human diseases. Here, we designed and performed a large-scale gain-of-function screen in Xenopus embryos aimed at identifying new regulators of MAPK/Erk, PI3K/Akt, BMP, and TGF-β/Nodal signalling pathways. Our gain-of-function screen is based on the identification of gene products that alter the phosphorylation state of key signalling molecules, which report the activation state of the pathways. In total, we have identified 20 new molecules that regulate the activity of one or more signalling pathways during early Xenopus development. This is the first time that such a functional screen has been performed, and the findings pave the way toward a more comprehensive understanding of the molecular mechanisms regulating the activity of important signalling pathways under normal and pathological conditions.

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Species referenced: Xenopus laevis
Genes referenced: bmp4 cer1 ctrl fbxo43 foxh1.2 mapk1 nodal nodal1 pik3ca prkaca smad1 smad2 wnt8a

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	Figure 2. Kinetics of the activation of signalling molecules during early Xenopus development.X. laevis embryos were collected at the time indicated and subjected to Western blot analysis. Membranes were probed with anti-phospho-Smad1/5/8 (pSmad1) antibody for monitoring BMP activity, anti-phospho-Smad2 (pSmad2) antibody for TGF-β/Nodal signalling, anti-phospho-Erk (pErk) for MAPK/Erk signalling and anti-phospho-Akt (pAkt) for PI3K/Akt signalling. Anti- Smad2, anti- Akt, and anti-Erk were used as loading controls to ensure all lanes have been loaded equally.
	Figure 3. Flowchart of the experimental procedure of the screen.A X. tropicalis library of unique, full-length clones has been established based on sequence comparison and clustering of over 1,220,000 ESTs, and rearrayed in a 96-well plate format. Pools of 8 mRNAs were prepared from pooled bacteria culture and in vitro transcription. Then in vitro transcribed mRNA pools were injected into fertilized X. laevis embryos at 1–2 cell stage. After microinjection, injected embryos were collected at stage 8 (blastula), stage 10.5 (gastrula), and stage 14 (neurula). Protein extracts from embryos were loaded onto SDS-PAGE for subsequent Western blot analysis. Antibodies used include anti-phospho-Smad1/5/8, anti-phospho-Smad2, anti-phospho-Akt, and anti-phospho-Erk. Once a potential active pool was identified, the pool was de-convoluted and single molecule injection was performed to identify the active molecule.
	Figure 4. Proof of principle of the screen.(A) 12 pools, each one with one clone of known activity, were selected from the full-length EST library and injected into embryos as described. Protein extracts from collected embryos were subjected to Western blot using indicated antibodies to observe phosphorylation changes of specific signalling molecules at blastula and gastrula stages. Note the reduction of phospho-Smad2 activity at gastrula stage on pool 2, and reduction of phospho-Smad1/5/8 activity on pool 11. (B) De-convolution of pool 2. cerberus is identified as a negative regulator of Smad2 (pSmad2, lower panel) but not of Smad1 (pSmad1, upper panel) phosphorylation at gastrula stage. (C) De-convolution of pool 11. wnt8a is identified as a negative regulator of Smad1/5/8 phosphorylation (pSmad1, middle panel) and activator of Wnt signalling (pLRP6, upper panel) at gastrula stage. UI: uninjected.
	Figure 5. Examples on identification and de-convolution of active regulators.(A–B) identification of the MAPK/Erk activator fbxo43 (erp1). (A) Western blot of stage 8 embryos injected with 12 pools (01–12) derived from plate #01 and 7 pools (01–07) from plate #02 and probed with anti-phospho-Erk (pErk) antibody. The arrow indicates increased Erk phosphorylation upon injection of mRNAs derived from plate #02, pool 07. (B) De-convolution of the above pool. Embryos injected with single RNAs were collected at stage 8 and uninjected lysate from stage 8 and stage 10.5 were used as negative and positive control of Erk phosphorylation respectively. The arrow indicates the active clone of TEgg009F05, identified in plate #2, column 08, row G. This clone was confirmed as the X. tropicalis fbxo43 (erp1) gene. (C–D) Identification of PI3K/Akt inhibitor prkaca. (C) Western blot of stage 10.5 embryos injected with 24 pools (01–12) derived from plate #09 and #10 and probed with anti-phospho-Akt (pAkt) antibody. The arrow indicates decreased Akt phosphorylation upon injection of mRNAs derived from plate #10, pool 02. (D) De-convolution of the above pool. mRNA synthesis, injection, and Western blot were performed as in (B) except that stage 10.5 embryos were used. The arrow indicates the active clone of TEgg046d13 is identified in plate #09, column 02, row E. This clone was later identified as encoding the X. tropicalis prkaca gene.
	Figure 6. Results of the screen.The 20 active clones identified during the screen were individually injected and analysed by Western blot to demonstrate their abilities to modulate the activities of different signalling pathways as shown in Table 3. Embryos were collected at the indicated stages, processed, and analysed by Western blot to assess the activities of Erk (pErk, panel A), Akt (pAkt, panel B), BMP (pSmad1, panel C), and TGFβ/Nodal (pSmad2, panel D). Control (ctrl) denotes uninjected embryos. Anti-Erk (Erk), anti-Akt (Akt), anti-Smad2 (Smad2) and anti-α-Tubulin (α-Tubulin) were used as loading controls.
	Figure 7. Whole-mount in situ hybridisation images on clones with regionalised expression patterns.For each clone the corresponding clone number and Xenopus gene symbol are shown. Vegetal view (stage 10.5 except for foxh1, which is side view); dorsal view (stage 15 and 20, posterior is up); lateral view (stage 30, anterior is to the left).
	Figure 1. Characterisation of phospho-specific antibodies.(A) Characterisation of anti-phospho-Akt (pAkt) and anti-phospho-Erk (pErk) antibodies, the PI3K/Akt inhibitor LY294002 and FGF inhibitor SU5402 were used to inhibit Akt and Erk phosphorylation in gastrula embryos, respectively. 1% DMSO was used to exclude any possible interference from the inhibitor solvent. Anti-Erk (Erk), anti-Akt (Akt) and anti-α-tubulin (α-tubulin) were used as loading controls. (B) Characterisation of anti-phospho-Smad1/5/8 (pSmad1) and anti-phospho-Smad2 (pSmad2) antibodies. The TGF-βRI inhibitor SB505124 was used to inhibit Smad2 phosphorylation in gastrula embryos; injection of wnt8a mRNA was used to inhibit bmp4 expression, thus preventing Smad1/5/8 phosphorylation. All inhibitors have been added at stage 6. 1% DMSO was used to exclude any possible interference from the inhibitor solvent. Smad2 and α-tubulin serves as internal controls to ensure equal loading in all lanes.

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