in the development and patterning of embryonic and adult tissues , secreted signaling molecules of the wnt , fgf , hedgehog , and transforming growth factor  ( tgf- ) families can act as morphogens to activate different transcriptional programs along a signaling gradient ( perrimon et  al . , 2012 ) . ideas of how morphogens impart spatial information have been dominated by the assumption that these molecules form concentration gradients by diffusion , inducing dose - dependent responses in the receiving field of cells . however , it is becoming increasingly clear that for some ligands , for example , hedgehog , wnt , and fgf , other mechanisms , such as short - range signaling activation , transcriptional feedback , and cellular rearrangements , underlie morphogen function ( alexandre et  al . regulation of nodal signaling in the zebrafish embryo has long  served as a paradigm for understanding how morphogens pattern tissues ( schier , 2009 ) . nodals are secreted ligands that belong to the tgf- superfamily of growth and differentiation factors . during vertebrate development , nodal is required for stem cell maintenance , specification of mesoderm and endoderm ( mesendoderm ) , and establishment of left - right asymmetry ( shen , 2007 ) . recent work has also suggested that nodal signaling is reactivated in advanced cancers , where it may be important for self - renewal of cancer stem cells ( wakefield and hill , 2013 ) . nodal ligands signal through serine / threonine kinase receptor complexes comprising two type i receptors ( acvr1ba [ taram - a ] ) , two type ii receptors ( acvr2a / b ) , and the co - receptor tdgf1 ( cripto / oep ) ( schier , 2009 , shen , 2007 ) . ligand binding activates the receptors , after which the type i receptor phosphorylates the intracellular signal transducers smad2 and smad3 , which then bind smad4 ( massagu , 2012 ) . these smad2/3-smad4 complexes accumulate in the nucleus , where , together with transcription factors such as foxh1 , mixer , and oct4 , they regulate gene transcription ( gaarenstroom and hill , 2014 ) . at zebrafish blastula stages , two nodal - related ligands , ndr1  ( squint ) and ndr2 ( cyclops ) , specify mesendoderm in marginal cells around the circumference of the embryo by inducing a smad2-smad4-foxh1-dependent transcriptional program ( feldman et  al . , 1998 , gritsman et  al . , 1999 ) . ndr1/2 are thought to form a signaling gradient by diffusion , extending up to about ten cell tiers from the margin ( dubrulle et  al . , 2015 , indeed , expression of presumed long - range nodal target genes such as ta ( ntla ) and fscn1a suggests low - level signaling up to ten cell tiers from the margin ( bennett et  al . , 2007 ) . this appears supported by bimolecular fluorescent complementation experiments ( harvey and smith , 2009 ) . however , other nodal target genes are expressed in up to five to six cell tiers from the margin , which coincides with nuclear accumulation of smad2-gfp fusion protein ( dubrulle et  al . , 2015 ) . importantly , other signaling pathways , such as bmp , wnt , and fgf , are also active at the margin , which can potentially co - regulate nodal target genes and thus contribute to their expression domains . formation of the nodal signaling domain at the correct time and of appropriate dimensions is thought to be controlled by a reaction - diffusion system ( meinhardt , 2009 , schier , 2009 ) . this model requires positive and negative feedback , which is provided by nodal - induced expression of both the ligands ndr1/2 and the antagonists lefty1 ( lft1 ) and lefty2 ( lft2 ) ( chen and shen , 2004 , cheng et  al . , 2004 ) . besides these feedback mechanisms , the model requires lft1/2 to be more diffusible than ndr1/2 ( mller et  al . , 2012 , schier and talbot , 2005 ) . these conditions are thought to allow ndr1/2 to activate signaling at the margin , whereas lft1/2 proteins would inhibit signaling in more distal cells . overexpression studies have shown that ndr1/2 and lft1/2 can differentially diffuse and that ndr1 , but not ndr2 , can diffuse over a distance to activate signaling ( chen and schier , 2001 , chen and schier , 2002 , mller et  al . , 2012 ) . however , the importance of diffusion of endogenous ndr1/2 remains unclear , as mesendoderm can develop normally in zygotic ndr1 mutants ( dougan et  al . , 2003 , feldman et  al . , 1998 , lim et  al . , in addition to the negative feedback provided by lft1/2 , nodal signaling is regulated by the mir-430/427/302 family of micrornas ( mirnas ) ( bassett et  al . , 2014 , choi et  al at blastula stages , the mir-430 family is the most abundant family of mirnas in the zebrafish . importantly , mir-430 regulates ndr1 , lft1 , and lft2 , but not ndr2 , and this is thought to dampen nodal signaling ( choi et  al . , 2007 ) . however , to what  extent mir-430s contribute to the formation of the nodal signaling domain is unknown . to develop a specific readout for endogenous nodal signaling , avoiding overexpression of any pathway components , we generated a transgenic zebrafish nodal reporter line . using this line combined with immunofluorescence for phosphorylated smad2 ( p - smad2 ) , we show that nodal signals exclusively in cells that express ndr1/2 , up to five to six cell tiers from the margin . this prompted us to revisit the mechanism underlying the formation of the nodal signaling domain . our data do not support the reaction - diffusion model , but instead , we propose that nodal activates signaling in a temporal window that is defined by a mir-430-mediated delay of lft1/2 translation . in this way , temporal information is converted into spatial information in the developing embryo . the range of activity of the nodal signaling pathway in the blastula margin has mainly been inferred from the expression of endogenous target genes , such as fscn1a and in particular ta ( ntla ) ( bennett et  al . , 2007 , gritsman et  al . , 1999 ) . however , in addition to nodal , fgf signaling is also known to regulate ta expression ( griffin et  al . , 1995 , rodaway et  al . , 1999 , schier and talbot , 2005 ) . genes encoding fgf ligands , such as fgf3 and fgf8a , are expressed in the margin ( figure  s1a ) and are known nodal targets ( mathieu et  al . , 2004 ) , suggesting that fgf signaling at the margin of blastula - stage embryos is downstream of nodal signaling ( rodaway et  al . , 1999 ) . this is clearly demonstrated by treating embryos with the nodal inhibitor sb-505124 ( hagos and dougan , 2007 ) , which results in a near complete loss of phosphorylated erk ( p - erk ) , a readout for fgf pathway activity ( dorey and amaya , 2010 ) ( figures 1a and 1b ) . given that both nodal and fgf signaling are active at the margin , we examined to what extent fgf signaling regulates endogenous nodal target genes , focusing on the expression of ta and fscn1a as examples of long - range genes and lft1 and lft2 as examples of short - range target genes ( bennett et  al . to inhibit fgf signaling , wild - type ( wt ) embryos were treated with the fgf receptor ( fgfr ) inhibitor su-5402 ( mohammadi et  al . , 1997 ) or were injected with mrna encoding a dominant - negative fgfr ( dnfgfr ) ( amaya et  al . , 1991 ) both treatments resulted in a reduction in the size of the expression domains of ta and fscn1a in the margin of 40%50% epiboly embryos , but not of lft1 or lft2 ( figure  1d ) . similarly , morpholinos ( mos ) against fgf3 and fgf8a resulted in a reduction of ta expression , but not of lft1 ( figure  s1b ) . qpcr on su-5402-treated 50% epiboly embryos confirmed the whole - mount in  situ hybridization ( wish ) results ( figure  1e ) , and as expected , inhibition of nodal signaling by sb-505124 led to reduction in expression of all four genes ( figure  1e ) . importantly , fgfr inhibition had no effect on c - terminal phosphorylation of smad2 ( p - smad2 ) or overall smad2 levels , demonstrating that nodal signaling is not affected by fgf signaling inhibition ( figure  1f ) . to quantitate the effect of inhibiting fgf signaling on the ta expression domain , we performed serial sectioning on ta - stained embryos at 40% epiboly . in control embryos , ta is expressed in an average of about 10 cell tiers from the margin , whereas expression was reduced to six cell tiers in su-5402-treated embryos ( figures 1 g and 1h ) . this indicated that ta expression beyond six cell tiers was due to fgf signaling and not directly dependent on nodal . in support of this idea , injection of increasing doses of fgf8a mrna into a maternal zygotic ( mz ) tdgf1 background confirmed that nodal signaling is not required for induction of ta by fgf , excluding a requirement for synergism between nodal and fgf signaling for ta expression beyond six cell tiers ( figure  s1c ) . this was further confirmed by the observation that inhibition of nodal signaling from the 16-cell stage resulted in the loss of both ta and lft1 expression , but when nodal signaling was inhibited from dome stage , only lft1 expression was severely reduced , whereas the expression of ta was unaffected ( figure  s1d ) . thus ta expression is not dependent on nodal activity after fgf signaling has been initiated . together these data demonstrate that fgf signaling regulates presumed long - range endogenous nodal target genes beyond six cell tiers . to exclusively monitor nodal signaling without inputs from other pathways an egfp reporter gene under the control of three foxh1 and smad binding sites , termed activin response elements ( ares ) ( germain et  al . , 2000 ) , was inserted into the zebrafish genome using tol2-mediated transgenesis ( tg[are : egfp ] ) ( figure  2a ) . we chose this reporter , which we have extensively characterized in a number of cell culture and developmental contexts ( germain et  al . , 2000 , inman and hill , 2002 , randall et  al . , 2004 ) , because foxh1 is the primary transcription factor required for immediate early nodal target gene expression ( pogoda et  al . , in addition , smad2 is the predominant receptor - regulated smad during blastula stages ( figure  s2a ) , and indeed mz deletion of smad2 results in a phenotype identical to mztdgf1 embryos ( dubrulle et  al . , 2015 ) . in four independent tg(are : egfp ) lines , wish for egfp mrna revealed that the reporter was activated in identical domains , excluding any effects caused by different integration sites of the transgene ( figures 2a and s2b ) . the egfp expression domains correspond to the expression domains of nodal ligands ( schier , 2009 ) and include the embryonic margin at blastula stages , axial mesoderm during gastrulation , and the left lateral plate mesoderm during somitogenesis ( figures 2a and s3 ) . incubation of developing tg(are : egfp ) embryos with sb-505124 resulted in a strong reduction of egfp staining ( figure  2b ) . this was also the case when foxh1 mos were injected or when the tg(are : egfp ) embryos were bred into a tdgf1 background ( figure  2b ) . the tg(are : egfp ) reporter is therefore specific for smad2-smad4-foxh1-mediated nodal signaling during blastula stages . to confirm the inducibility of the reporter gene , we exposed dissociated cells from tg(are : egfp ) blastula - stage embryos to increasing concentrations of recombinant nodal and measured gene expression by qpcr . egfp expression was induced at identical nodal concentrations compared with ta , suggesting equal sensitivity of the reporter compared with this target gene ( figure  2c ) . historically the sensitivity of nodal target genes , such as ta and noto ( flh ) , has been assessed by ectopically expressing nodal ligands in the animal pole of blastula - stage embryos and using wish to assay gene expression ( chen and schier , 2001 ) . using nodal - coated beads in such an assay , we found that the reporter was induced in a domain of similar size to that of ta and noto ( figure  2d ) . finally , we found no differences in the expression of downstream nodal genes in 40%50% epiboly embryos by qpcr and wish when we compared wt and tg(are : egfp ) embryos , demonstrating that the introduction of the transgene had no impact on nodal signaling ( figures s2c  s2e ) . in conclusion , the tg(are : egfp ) zebrafish line is a specific and sensitive biosensor for early nodal signaling . we next used the tg(are : egfp ) reporter line to determine how  nodal signaling is initiated in the embryo . at the 8-cell and 1,000-cell stages , no egfp mrna could be detected ( figure  2e ) , indicating that egfp mrna is not maternally contributed . at sphere stage , however , signaling cells are detected as a localized cluster of egfp - positive cells ( figure  2f ) . from 30% epiboly until the onset of gastrulation , nodal signaling is detected in the  entire margin , with a shallow staining gradient running from dorsal to ventral ( figure  2f ) . as expected , the overall expression of egfp correlated well with increasing levels of p - smad2 ( figure  2 g ) . we compared the spatial and temporal activation of nodal signaling in tg(are : egfp ) embryos with the expression of core components of the pathway ( figure  s3 ) . the genes encoding the receptor acvr1ba , the co - receptor tdgf1 , and transcription factors smad2 and foxh1 are ubiquitously expressed during blastula stages . the activation of signaling therefore depends exclusively on ligand expression ( figure  s3 ) . the discrete signaling domain at sphere stage likely corresponds to the future dorsal side of the embryo , given the known dorsal localization of maternal ndr1 mrna ( figures s3 and s4a ) ( gore et  al . we could show that ndr1 transcripts are polyadenylated before the maternal - to - zygotic transition ( figure  s4b ) , suggesting that maternal ndr1 is translated and may signal during blastula stages . this was demonstrated by knocking down the mix - like transcription factor mxtx2 , which is required for zygotic ndr1 and ndr2 expression in the yolk syncytial layer ( ysl ) ( fan et  al . , 2007 , hong et  al . , 2011 , xu et  al . , 2012 ) . tg(are : egfp ) mxtx2 morphants retained only a small , dorsal domain that expressed ndr1 , ndr2 , and egfp ( figure  s4c ) . loss of the ventrolateral expression of ndr1/2 in the blastoderm in mxtx2 morphants suggested that this ndr1/2 expression was initiated by nodal ligands secreted by the ysl and resulted from the ability of ndr1/2 to induce their own expression . indeed , embryos incubated with the nodal inhibitor sb-505124 from the 32-cell stage exhibited complete loss of expression of ndr1/2 in the blastoderm of 40% epiboly embryos , whereas expression of ndr1/2 in the ysl was not affected ( figure  s4d ) ( fan et  al . , 2007 ) . thus , maternally provided ndr1 activates nodal signaling in dorsal - most embryonic cells before the initiation of signaling in the entire margin by ndr1/2 synthesized in the ysl ( figure  s4e ) . the tg(are : egfp ) zebrafish line provides an ideal tool to investigate the dimensions of the nodal signaling domain at the margin . to initially investigate whether there was any nodal signaling beyond the ligand - expressing domain , we performed double fluorescent wish for ndr2 and egfp in 30% epiboly embryos , when the nodal signaling domain is expanding ( dubrulle et  al . , 2015 , harvey and smith , 2009 ) . although individual signaling cells were occasionally observed directly adjacent to ndr2-expressing cells , no egfp - positive cells were detected further beyond the ligand expression domain ( figure  s5a ) . to  determine the extent of nodal signaling more rigorously , 40%50% epiboly tg(are : egfp ) embryos were stained for egfp , ndr1 , ndr2 , lft1 , and lft2 expression , sectioned , and quantitated ( figures 3a and 3b ) . the expression of egfp , ndr1 , ndr2 , and lft1 was limited to an average of about five cell tiers , whereas lft2 expression was detected in only two to three cell tiers from the margin . to confirm that these findings were not due to a lack of sensitivity of the wish , we analyzed the expression of sox3 , which is repressed by nodal signaling in the margin ( bennett et  al . significant repression was seen in up to six cell tiers from the margin ( figure  3a ) . importantly , staining for all induced nodal target genes , including egfp , was consistently stronger in a cluster of cells directly proximal to the ysl , where signaling originates ( figure  3a ) . to corroborate the data obtained from the sections , we used a direct readout of the nodal pathway , fluorescent immunostaining of whole - mount 50% epiboly embryos with an antibody against p - smad2 . for comparison , we also stained for p - erk . nuclear p - smad2 staining was observed exclusively in the margin and , as expected , in a smaller domain than the p - erk staining ( figure  3c ) . to quantitate the size of the p - smad2-positive  domain , we imaged the embryos at the margin , used metamorph software to generate normalized nuclear staining intensities , and measured the distance of each nucleus from the margin in control- and sb-505124-treated embryos . p - smad2 staining above background was found up to five to six cell tiers ( 8090  m ) from the margin in a steep gradient , which was abolished in sb-505124-treated embryos ( figures 3d , 3e , and s5b  s5d ) . the staining was strongest in the nuclei of cells nearest the ysl . taken together with our observations from the tg(are : egfp ) reporter line , these data demonstrate that nodal signaling occurs in five to six cell tiers from the margin , closely mirroring the expression of the ligands . our data demonstrate that the nodal ligands and antagonists are co - expressed at the margin of late blastula - stage embryos , and moreover , nodal activity is restricted to these cells . first , if the ligands and antagonists are co - expressed , how does signaling occur at all ? second , given that all cells are competent to signal at blastula stages and nodal signaling induces the expression of the ligands , why does signaling not spread throughout the embryo ? to address these questions , we first determined to what extent lft1/2 regulate nodal signaling in early and late blastula - stage embryos . the lft1/2 proteins are known to inhibit signaling by sequestering tdgf1 and possibly also nodal itself ( chen and shen , 2004 , cheng et  al . , 2004 ) . as expected , injection of lft1/2 mos led to an expansion of the nodal signaling domain at 50% epiboly , which was confirmed by qpcr ( figures 4a and 4b ) . at dome stage , however , there was no increase in the size of the nodal signaling domain , suggesting that lft1/2 do not regulate nodal signaling at early stages . although there is an offset in the appearance of ndr1/2 and lft1/2 mrna due to the presence of maternal ndr1 transcripts , the lack of an early role for lft1/2 is not explained by an absence of lft1/2 mrna at sphere and dome stages ( figures 4c and s3 ) . we hypothesized therefore that a delay in translation of the lft1/2 proteins could account for the inability of lft1/2 to regulate nodal signaling at dome stage . both lft1 and lft2 transcripts were readily polyadenylated by sphere stage ( figure  4d ) , suggesting that lack of polyadenylation could not account for any delay in lft1/2 translation . to measure endogenous protein levels directly , we raised polyclonal antibodies for lft1 and lft2 and thoroughly characterized them ( figure  s6 ) . although both antibodies recognized their corresponding target protein when overexpressed ( figure  s6b ) , only the lft1 antibody was able to detect endogenous protein , so we focused on this family member . the major band detected by western blot corresponding to endogenous lft1 migrated by sds - page with a molecular weight of 40 kd ( figure  s6c ) . mutation analysis indicated that this product arose from cleavage at the first furin cleavage site ( marked as c1 in figure  s6a ) , and we could demonstrate that this product was active ( figures s6d  s6f ) . the same 40 kd band was also detected in embryos injected with ndr1 mrna ( figure  s6 g ) . levels of endogenous lft1 were barely detectable by western blotting at dome stage and 30% epiboly but increased at 50% epiboly ( figures 4e and 4f ) . thus , endogenous lft1 protein levels remain low until 50% epiboly , despite readily detectable mrna levels at all these time points . this suggested that the lack of an early role for lft1/2 could be due to low protein abundance and led us to hypothesize that repressed lft1/2 translation creates a window of opportunity for nodal signaling to become established . during blastula stages , the mir-430 family of mirnas have been reported to block translation , without affecting polyadenylation ( bazzini et  al . we reasoned therefore that the activity of mir-430 could be responsible for the repressed translation of lft1/2 proteins to create a temporal window for nodal to activate signaling . mir-430 pri - mirna is expressed in the nuclei of all cells in the blastoderm , immediately after activation of zygotic transcription ( figure  5a ) . we also confirmed a ubiquitous expression pattern for mature mir-430a and mir-430b at 50% epiboly ( figure  5b ) and demonstrated that mature mir-430a and mir-430b are directly processed upon expression ( figure  5c ) . mir-430c was not detected by either wish or northern blotting , but rna sequencing data demonstrated that mir-430c is much less abundant than mir-430a and mir-430b ( unpublished data ) . thus , the mir-430 family is abundant , ubiquitously expressed , and readily processed during mid to late blastula stages . to determine the role of mir-430 in the regulation of lft1/2 protein translation , we designed three mos that prevented processing of mature mir-430a , mir-430b , and mir-430c . injection of these mos into one - cell - stage embryos resulted in a phenotype resembling mz dicer mutants at 22 hpf ( figure  s7a ) ( giraldez et  al . , 2005 ) . furthermore , they abolished mir-430a staining at 50% epiboly and reduced mir-430a , mir-430b , and mir-430c expression , as determined by qpcr , by 89% ( figures s7b and s7c ) . co - injection of mir-430 mos with a gfp reporter containing either three mir-430 binding sites or a gfp reporter with the lft2 3utr , which contains a single mir-430 binding site , resulted in increased translation compared to control mos ( figure  s7d ) . we next injected equal amounts of control or mir-430 mos and performed western blotting for p - smad2 and lft1 at several blastula stages . in control mo - injected embryos , lft1 protein was not detectable until 30% epiboly and increased at 50% epiboly , as observed for lft1 protein expression in wt embryos ( compare figure  4e with figure  5d ) . importantly , this was accompanied by a gradual increase of p - smad2 over time . in contrast , injection of mir-430 mos led to premature translation of lft1 from dome stage , and this coincided with lower overall accumulation of p - smad2 ( figure  5d ) . interestingly , the level of lft1 protein in dome - stage mir-430 morphants was similar to the maximal level of lft1 protein measured at 50% epiboly in control mo - injected embryos , suggesting that there may be a threshold level of lft1 that is inhibitory . to determine whether reduced signaling in the mir-430 morphants was due to premature translation of lft1/2 and to investigate the spatial consequences for nodal signaling of the loss of mir-430 , we injected mir-430 mos and/or lft1/2 mos into tg(are : egfp ) embryos and assayed nodal activity ( egfp ) and ndr1 levels at 30% epiboly . injection of lft1/2 mos alone resulted in a modest increase in egfp and ndr1 staining ( figure  5e ) , whereas injection of mir-430 mos led to a reduction in egfp staining in the blastoderm , consistent with the inhibition of the nodal signaling pathway we observed using p - smad2 levels as a readout ( figure  5d ) . when lft1/2 mos and mir-430 mos were co - injected , signaling was activated in the entire blastoderm , and this was accompanied by a similar expansion of ndr1 expression ( figure  5e ) . importantly , this spreading of egfp staining in lft1/2 and mir-430 mo - co - injected embryos was due to nodal signaling , because it was completely blocked by incubating double morphants with sb-505124 ( figure  5f ) . the further spreading of signaling following combined knockdown of lft1/2 and mir-430 is readily explained by the regulation of ndr1 translation by mir-430 in the absence of lft1/2 ( see figure  7a ) ( choi et  al . , 2007 ) . together , these experiments demonstrate that mir-430 delays lft1/2 translation to create a temporal window for nodal to activate signaling . our data demonstrate that a temporal window for nodal signaling activation determines the size of the nodal signaling domain and predict that once lft1/2 levels reach a certain threshold , nodal signaling is unable to spread to adjacent cells . we therefore tested if blocking signaling activation by recombinant mouse lefty1 ( mlefty1 ) is dose dependent in dissociated embryonic cells . a 5-fold excess ( calculated by mass ) of mlefty1 over human recombinant nodal led to a near complete inhibition of signaling activation as read out by western blotting for p - smad2 and endogenous zebrafish lft1 , which monitors the transcriptional output of the pathway ( figure  6a ) . thus , lft1/2 proteins can reach an inhibitory concentration at which signaling can no longer be activated . next we determined if duration of exposure to nodal directly corresponds to increasing levels of signaling . when blastula - stage cells were exposed to 50  ng / ml nodal and then inhibited with a blocking concentration of 500  ng / ml mlefty1 at different time points , p - smad2 and endogenous lft1 levels were indeed proportional to the duration of ligand exposure ( figure  6b ) . finally , we investigated how rapidly signaling is blocked when lft1/2 levels reach inhibitory concentrations . this is a crucial issue , as we observe that nodal signaling in  vivo ( as read out by egfp , ndr1 , ndr2 , lft1 , and lft2 ) is sustained in the margin for several hours after lft1 levels reach an inhibitory concentration at around 50% epiboly ( figure  s3 ) . dissociated embryonic cells were therefore exposed to nodal for 1  hr , and then signaling was inhibited by addition of mlefty1 for 2  hr . we observed that p - smad2 levels decreased slowly , compared with the rapid termination of signaling with sb-505124 ( figure  6c ) . this demonstrated that although signal activation is blocked by lefty , nodal signaling is sustained for some time , presumably because of continued signaling from internalized receptor complexes in early endosomes ( jullien and gurdon , 2005 , vizn et  al . , 2013 ) . in conclusion , our data show that activation of nodal signaling in blastula - stage cells can occur until lft1/2 levels reach inhibitory concentrations . moreover , the levels of p - smad2 , and as a result transcription , are proportional to the duration of signal activation . this can be maintained for some time after inhibitory lft1/2 concentrations are reached , while no new signaling is activated . here we describe a specific and sensitive nodal reporter line that  has enabled us to visualize endogenous nodal signaling in developing zebrafish embryos , without overexpression of any pathway components . we show that signaling is initiated on the dorsal side because of maternally provided ndr1 . ventral and lateral signaling arises as a result of ndr1/2 expression in the ysl , which then spreads toward the animal pole as a result of autoregulation . nodal signaling in the margin reaches a maximum of six cell tiers , which we demonstrate by p - smad2 immunostaining and reporter activity . we find no evidence of signaling beyond the cells that express the ligand , and moreover , these same cells additionally express the nodal antagonists lft1/2 . spreading of presumed long - range nodal target genes , such as ta , that are activated beyond the ligand expression domain , our data support a model whereby a temporal window for nodal signaling activation dictates the dimensions of the  nodal signaling domain ( figure  7 ) . thus , temporal information is translated into spatial information in the developing embryo . the crucial determinant of the temporal window is the delayed translation of the lft1/2 proteins , which is mediated by mir-430 . in addition , maternally provided ndr1 transcripts and the production of ndr1/2 by the ysl allow nodal signaling to be initiated in the blastoderm , before transcription of lft1/2 ( figure  7c ) . because of positive feedback , the blastoderm cells produce more ndr1/2 , while lft1/2 levels remain relatively low because of the ubiquitous synthesis of mir-430 . in these conditions , nodal signaling can be activated in neighboring cells until extracellular lft1/2 levels reach inhibitory concentrations . therefore , the duration for which lft1/2 levels are repressed dictates the size of the nodal signaling domain . how the repressive action of mir-430 is lifted at 50% epiboly to allow lft1/2 translation is not yet known and requires further investigation . although inhibitory lft1/2 levels prevent further activation of signaling , and hence additional spreading of nodal signaling , cells already responding to nodal will continue to signal for several hours , because this occurs from internalized receptors that are refractory to lft1/2 inhibition . a consequence of our proposed mechanism is that cells directly adjacent to the ysl activate nodal signaling for the longest duration . this likely explains the more intense p - smad2 staining in these cells relative to those further from the margin . we therefore propose that nodal signaling at the margin at blastula stages is dictated by an interplay among ligand , ligand antagonist , and a mirna , with a differential in timing between ligand and antagonist production being the key determining factor . the size and shape of the nodal signaling gradient had previously been thought to be regulated by a reaction - diffusion system ( meinhardt , 2009 , mller et  al . , 2012 , schier , 2009 ) . here we propose an alternative mechanism whereby the size of the domain is dictated by the delay in lefty translation . although the nodal / lefty ligand / antagonist pair has many features of a reaction - diffusion system , we have uncovered one aspect that is incompatible . in reaction - diffusion models , which were originally conceived as pattern - forming chemical reactions , a homogeneous distribution of activator and inhibitor are unstable , and a local elevation of activator initiates formation of a gradient ( meinhardt , 2009 ) . integral to this model is the ability of the diffusing antagonist to immediately inhibit activator function at a distance . for nodal and lefty , this can not happen , because once nodal signaling is activated it occurs from internalized receptors and is therefore insensitive to lefty inhibition , except over prolonged time frames . our data suggest that spatially graded activity of the nodal signaling pathway is mainly the result of different durations of exposure to ndr1/2 over time , as opposed to exposure to different concentrations . we observe two graded signaling domains in tg(are : egfp ) embryos that are both explained by timing of ligand exposure . during blastula stages , a shallow signaling gradient runs from dorsal to ventral . this is readily explained by the fact that dorsal cells are exposed to nodal for a longer period than ventrolateral cells , because maternally provided ndr1 signals dorsally before ndr1/2 produced in the ysl induces signaling in the blastoderm margin . in addition , we found a clear vegetal - to - animal gradient within the ligand - expression domain using p - smad2 immunostaining in late blastula - stage embryos , and we also observed that cells directly adjacent to the ysl expressed higher levels of all nodal target genes , including the egfp reporter gene , reflecting higher levels of signaling . again , cells directly adjacent to the ysl are exposed to ndr1/2 for the longest period of time . the importance of duration of exposure is further supported by our ex  vivo experiments with dissociated embryonic cells . the long - term functional consequences of this were demonstrated in previously published work , which linked cell fates to the duration of exposure to nodal signals ( hagos and dougan , 2007 ) . thus , our model explains how concentration and duration of signaling can be translated into positional information . finally , the importance of timing of signaling activation also rationalizes the normal development of ndr1 mutants ( feldman et  al . , 1998 , heisenberg and nsslein - volhard , 1997 , lim et  al . , 2013 ) . this is explained by the fact that ndr2 compensates for the lack of ndr1 , but its expression is delayed , because there is no maternal ndr2 . from the sectioning of tg(are : egfp ) embryos and immunostaining for p - smad2 , it is clear that smad2-smad4-foxh1-dependent nodal signaling is confined to 56 cell tiers from the margin , which could imply that all mesendodermal cells arise from this domain . this finding seems to contradict lineage - tracing studies that show that some mesodermal precursors are located up to 12 cell tiers away from the margin ( e.g. , see dougan et  al . although these lineage - tracing studies have provided valuable insight into the overall spatial distribution of mesendoderm precursors , they can not themselves determine the extent of nodal signaling . this is because although nodal signaling is required for mesendoderm formation , not all cells that become mesendoderm have necessarily experienced nodal signaling directly . our understanding of how morphogens activate graded signaling in tissues has been dominated by the pre - molecular era assumption that secreted ligands diffuse from a source to form concentration gradients , and this assumption has naturally progressed into the formulation of models that include diffusion as a  major determinant in patterning by morphogens . although in some contexts , such as the establishment of left - right asymmetry , nodal can act at long range ( shiratori and hamada , 2014 ) , the work presented here shows that the formation of the nodal signaling domain at the blastula margin is explained by short - range signaling activation , signaling dynamics , and transcriptional / translational regulation . human nodal ( 3218-nd / cf ; r&d ) was dissolved in 4  mm hcl at 100  g / ml , aliquoted in non - stick tubes , and used at 40  ng / ml ( unless stated otherwise ) without freeze - thawing . recombinant mlefty1 was dissolved according to the manufacturer s instructions ( 994-lf / cf ; r&d ) . the inhibitors sb-505124 ( 3263 ; tocris bioscience ) and su-5402 ( 572631 ; calbiochem ) were dissolved in dmso and used in embryos at 50 and 10  m respectively . in dissociated embryonic cells , up to 1,000 embryos were dechorionated using 2  mg / ml pronase ( 11459643001 ; roche ) in 10  ml e3 medium . the dechorionated embryos were washed extensively in e3 medium and once with calcium - free ringers buffer to remove the pronase ( link et  al . , 2006 ) . the embryos were manually disrupted in calcium - free ringers buffer using a p200 pipette in 6  cm bacterial dishes , collected by centrifugation at 1,000   g for 5  min , and then resuspended at 50 embryos / ml in leibovitz s l15 medium ( 11415 - 064 ; gibco ) supplemented with 3% fetal bovine serum ( fbs ) . cells were plated in 24-well tissue culture plates coated with poly - l lysine ( p4707 ; sigma ) and allowed to attach for 30  min . embryos were fixed in 4% paraformaldehyde in pbs overnight , dehydrated to 100% methanol , and stored at 20c until processing . for whole - mount immunofluorescence , embryos were rehydrated to pbs and incubated in cold acetone at 20c for 20  min . blocking and antibody incubations were performed in 10% fbs and 1% triton x-100 in pbs , and washes were performed in pbs/1% triton . the following primary antibodies were used : -p - smad2/3 ( 8828 ; cell signaling technology ) and -p - erk ( m8159 ; sigma ) . note that because of the lack of smad3 at blastula stages , we solely detect p - smad2 with the -p - smad2/3 antibody . dapi was used to stain nuclei , and images were acquired on a zeiss lsm 780 confocal microscope . for quantification of p - smad2 intensity in deep cells , metamorph software ( molecular devices ) was used to generate p - smad2 to dapi ratios from at least three single optical slices per embryo , in three individual dmso or sb-505124 treated , 50% epiboly embryos . care was taken not to select mitotic or overlapping nuclei or nuclei from the enveloping layer . to measure the distance of a nucleus to the margin , a line was drawn laterally at the vegetal edge of the margin , and the distance to the nucleus perpendicular to the margin was measured using the metamorph software . to normalize the staining intensity for each embryo , the average ratio of an area further than 11 cell tiers from the margin ( > 165  m ) was subtracted from each measurement . the data were divided in 15  m bins , representing the average size of a deep cell at 50% epiboly ( unpublished data ; dubrulle et  al . , the averages of the binned data for each cell tier , comparing dmso and sb-505124 treatment , were used for testing for statistical significant differences using paired t tests with a 95% confidence interval . all the zebrafish work was carried out under a uk home office license under the animals ( scientific procedures ) act 1986 . the license underwent full ethical review and approval by the cancer research uk london research institute animal ethics committee . , c.h . , and m .- c.r . performed the experiments and analyzed the data with the help of r.k.s .