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A changing morphogen gradient is interpreted by continuous transduction flow

Wellcome/CRC Institute, Tennis Court Road, Cambridge, CB2 1QR, and Department of Zoology, University of Cambridge, UK

View larger version (44K): [in a new window]   Fig. 1. Cells memorise activin concentration for at least a few hours. (A) Design of the experiment; results shown in B. Dissociated animal cap cells were treated with a concentration of 4 ng/ml of activin for 10 minutes, either at stage 8.5 or at stage 9, washed to remove the unbound activin, and cultured for various lengths of time (from 1 hour 20 minutes up to 6 hours). The double arrows indicate a 10 minute exposure to activin and the black lane represents the culture time length. (B) Cells remember their activin treatment for over 3 hours. RNAse protection analysis of Xbra expression. WE, whole embryo. Un, untreated cells. (C) Prediction of the Eomes:Apod expression ratio over time, assuming that accumulation of transcripts increases linearly with time, that degradation of transcripts is 30% per hour, that Apod and Eomes mRNAs stabilities are similar, and that Apod transcription is initiated at a quarter the activin concentration required to initiate Eomes transcription. (D) RNAse protection analysis of Apod and Eomes expression. Dissociated animal cap cells were treated with a concentration of 4 ng/ml of activin for 10 minutes, at stage 8.75, washed to remove the unbound activin and cultured for various lengths of time (from 1 hour 20 minutes up to 4 hours). (E) Summary of three separate experiments.The Eomes:Apod expression ratio does not change with time from activin treatment. Dissociated animal cap cells were treated with either of 4 ng/ml or 10 ng/ml of activin for 10 minutes at stage 8.5-9, washed to remove the unbound activin, and cultured for various lengths of time (from 40 min up to 4 hours 40 minutes). The expression of Apod and Eomes was assayed by RNase protection and quantitated. (F) Cells memorise activin concentration in the absence of protein synthesis. Dissociated animal cap cells pretreated (+) or not (-) with cycloheximide for 1 hour, were then treated with a concentration of 4 ng/ml of activin for 10 minutes at stage 9, washed to remove the unbound activin, cultured for 3 hours and gene expression was assayed by RNase protection. The Eomes:Apod expression ratio is shown on the right. (G) Chordin activation is repressed by cycloheximide. Animal caps, pretreated or not with cycloheximide for 1 hour, were then cultured with a concentration of 4 ng/ml of activin until stage 10.5 and gene expression was assayed by RNase protection.

View larger version (52K): [in a new window]   Fig. 2. Modified forms of Smad2 are valid markers of concentration-dependent activin transduction. (A) Schematic representation of the Myc-Smad2, GST-Smad2 and GFP-Smad2 fusion constructs. (B) Myc-Smad2, GST-Smad2 and GFP-Smad2 are almost equally efficient at inducing Eomes and Xbra. Each embryo was injected with the indicated amount of Myc-Smad2, GST-Smad2 or GFP-Smad2 mRNA. Animal caps were dissected at stage 8.5, cultured until stage 10.5 and gene expression was assayed by RNase protection. WE, whole embryo. (C) Induction of Xbra by injection of untagged Smad2 mRNA. Reproduced, with permission, from Shimizu and Gurdon (Shimizu and Gurdon, 1999). (D) GFP-Smad2 accumulates in the nucleus in response to activin signalling. Dissociated animal cap cells from GFP-Smad2-injected embryos were treated with activin for 15 minutes, and loaded onto a fibronectin substrate. When control embryos reached stage 10-10.5, the cells were observed by confocal microscopy.

View larger version (39K): [in a new window]   Fig. 3. Phosphorylation and accumulation of Smad2 occur rapidly and in proportion to activin concentration. (A) Phosphorylation of Smad2 can be detected 20 minutes after activin treatment. Dissociated animal cap cells from GST-Smad2-injected embryos were treated with activin (3 ng/ml) for 15 minutes, reaggregated, and cultured for the indicated length of time. GST pulled down proteins from the cell lysates were resolved on SDS-PAGE and transferred to nitrocellulose. The membrane was first blotted with an anti-phosphoserine antibody (top panel) to detect the phosphorylation of Smad2 and then blotted with an anti-GST antibody (bottom panel) to visualise total GST-Smad2. (B) GFP-Smad2 quickly enters the nucleus in cells treated with activin. Dissociated animal cap cells from GFP-Smad2-injected embryos were loaded onto a fibronectin substrate. When control embryos reached the indicated stage, the activin (5 ng/ml) was added to cells directly on the slides, and movement of GFP-Smad2 was observed by confocal microscopy in real time. (C) The phosphorylation of Smad2 is activin dose dependent. Dissociated animal cap cells from Myc-Smad2-injected embryos were treated with increasing doses of activin for 15 minutes and cultured in medium supplemented with 32P-orthophosphate, until stage 10.5. Cells lysates were subjected to immunoprecipitation with anti-Myc antibody. Immunoprecipitated proteins were resolved on SDS-PAGE and transferred to nitrocellulose. The phosphorylation of Smad2 was detected by autoradiography (top panel). The membrane was then blotted with an anti-Myc antibody (bottom panel) to visualise total Myc-Smad2. The Smad2 phosphorylation was quantitated and the values plotted. (D) The nuclear accumulation of Smad2 is activin concentration related. Dissociated animal cap cells from GFP-Smad2-injected embryos were treated with increasing concentrations of activin at the indicated stages, and loaded onto a fibronectin substrate. Thirty minutes later, the localisation of GFP-Smad2 was analysed by confocal microscopy. The nuclear GFP-Smad2 concentration was quantitated using the public-domain NIH Image program (Teleman and Cohen, 2000) and the values are shown at the bottom.

View larger version (35K): [in a new window]   Fig. 4. An activated nuclear Smad2 pool is inherited through mitosis and persists for at least 4 hours. (A) The absolute increase in cell number, during the activin competence period, as a function of stage is depicted. (B) Nuclear Smad2 is relocalised to the nucleus after mitosis. Confocal microscopy observation, in real time, of GFP-Smad2 localisation in activin treated cells undergoing mitosis. The nuclear GFP-Smad2 concentration was quantitated and the values are shown on the right. (C) Transient activin treatment leads to the formation of a stable pool of phosphorylated Smad2. The experiment was designed as described in Fig. 3A. (D) GFP-Smad2 is localised in the nucleus for several hours after activin treatment. Dissociated animal cap cells from GFP-Smad2-injected embryos were treated with activin and loaded onto fibronectin substrates. The localisation of GFP-Smad2 was observed in the same cell, over a period of 4.5 hours, by confocal microscopy in real time. The quantitation of the nuclear GFP-Smad2 concentration is shown on the right. (E) Confocal microscopy observation, in real time, of GFP-Smad2 distribution in activin treated cells cultured on a fibronectin substrate. Yellow arrows indicate cells containing a small pool of GFP-Smad2, and red arrows cells with a bigger pool of GFP-Smad2.

View larger version (24K): [in a new window]   Fig. 5. Nuclear Smad2 concentration is maintained by continuous degradation and nuclear entry. (A) Smad2 has a 2-3 hour half-life in activin-treated cells (graph). Dissociated animal cap cells were subjected to a pulse-chase metabolic labelling (with [35S]methionine/cysteine), treated or not with activin, and Smad2 was immunoprecipitated with an anti-Smad2 antibody. The specificity of immunoprecipitation was confirmed by using protein-A-sepharose alone (data not shown). (B) Activated Smad2 has a 1-1.5 hour half-life (graph). Dissociated animal cap cells were subjected to a pulse-chase metabolic labelling (with [32P]orthophosphate), treated with activin, and Smad2 was immunoprecipitated with an anti-Smad2 antibody. Un, untreated. (C) Phosphorylation of Smad2 is detected long after activin treatment. Dissociated animal cap cells were treated with activin for 15 minutes and cultured in medium supplemented with [32P]orthophosphate either just after or 1.5 hours after activin treatment. Smad2 was immunoprecipitated with an anti-Smad2 antibody. (D) GR-GFP-Smad2 fusion construct. (E) Dexamethasone treatment can induce nuclear Smad2 localisation, several hours after activin has been removed. Dissociated animal cap cells from GR-GFP-Smad2-injected embryos were treated with activin for 15 minutes, extensively washed, loaded onto a fibronectin substrate before control embryos reached stage 9. Three hours after stage 9, GR-GFP-Smad2 release was induced by addition of dexamethasone and GR-GFP-Smad2 localisation observed by confocal microscopy in real time.

View larger version (22K): [in a new window]   Fig. 6. The Smad2 flow volume is used by the cells to interpret changing morphogen concentration. (A) Cells can switch from a low dose to a high dose gene response, but not from a high to a low dose gene response. RNase protection analysis of Apod and Eomes expression. Dissociated animal cap cells were treated either with 1 ng/ml of activin for 15 minutes (track 1), or with 4 ng/ml for 15 minutes (track 2), or with 1 ng/ml for 15 minutes and with 4 ng/ml for 15 minutes (track 3), or with 4 ng/ml for 15 minutes and with 1 ng/ml for 15 minutes (track 4). Cells were extensively washed after each dose of activin and cultured until control embryos reached stage 10.5. WE, whole embryo. UN, untreated cells. (B) Design of the experiment; results shown in C. Dissociated animal cap cells from GFP-Smad2-injected embryos were treated with a first dose of activin (1 or 4 ng/ml) for 15 minutes and loaded onto fibronectin substrates. At time T0, a first photo of the cells is taken and the cells receive a second dose of activin (4 or 1 ng/ml) or only buffer. At T0 + 30 minutes, a second photo of the same cells is taken. (C) The amount of nuclear Smad2 increases in response to higher concentrations of activin, but does not decrease with lower concentrations. GFP-Smad2 localisation is observed by confocal microscopy in real time. The nuclear GFP-Smad2 concentration was quantitated using the public-domain NIH Image program and the values are plotted on the bottom graph.

View larger version (19K): [in a new window]   Fig. 7. (A) The cytoplasmic pool of Smad2 is big enough to supply the Smad2 flow for several hours. Dissociated animal cap cells, pretreated or not with cycloheximide for 1 hour, were then treated with a concentration of 4 ng/ml of activin for 15 minutes at stage 9, washed to remove the unbound activin, cultured for 3 hours and the total amount of Smad2 was determined by western blot, using an anti-Smad2 antibody. (B) Quantitation of the total amount of Smad2 from the experiment shown in A, using the public-domain NIH Image program.

View larger version (30K): [in a new window]   Fig. 8. The receptor complex remains activated after a short exposure to activin. (A) Gene expression is blocked by serine/threonine kinase inhibitor H7. RNase protection analysis for Apod, Eomes and Xbra expression, in activin-induced cells treated with the indicated concentration of inhibitor H7. WE, whole embryos. (B) H7 inhibits phosphorylation on the C-terminal serine of Smad2. Dissociated animal cap cells from GST-Smad2-injected embryos were treated with activin for 15 minutes, reaggregated and cultured minus or plus H7 inhibitor, at the indicated concentration, until stage 10.5. GST pulled down proteins from the cell lysates were resolved on SDS-PAGE and transferred to nitrocellulose. The membrane was first blotted with an anti-phosphoserine antibody (top panel) to detect the phosphorylation of Smad2 and then blotted with an anti-GST antibody (bottom panel) to visualise total GST-Smad2. (C) H7 blocks gene activation by a constitutively active type I receptor (Alk4*), whereas gene activation by a constitutively phosphorylated Smad2 (Smad2*) is not affected by H7. RNase protection analysis for Apod, Eomes and Xbra expression, in animal cap cells from Smad2*- or Alk4*-injected embryos, treated with the indicated concentration of inhibitor H7. WE, whole embryos. (D) Delayed exposure to the inhibitor H7. Inhibitor was added at the times indicated by the red lines (left), and has progressively less effect to gene expression the later it was added to cells. RNase protection analysis for Apod, Eomes and Xbra expression. WE, whole embryos. (E) Quantitation of the gene expression over FGF-R ratio from the experiment shown in D.