BACKGROUND
the complex process of embryogenesis is directed by the regulation of signalling pathways that are achieved in part by the activity of a variety of secreted ligands.
thus understanding the temporal and spatial activity of signalling peptides is key to determining the role for these factors in controlling cellular fates.
for example, fibroblast growth factors , a family of secreted glycoproteins, perform crucial functions that include the establishment of embryo polarity, the formation of organizing centres, and the induction of limb outgrowth  <cit> .
these ligands are expressed in discrete domains during development and their actions are restricted to cells that express integral membrane proteins that can bind fgfs  <cit> .
the fgf receptors  are members of the receptor tyrosine kinase  class of transmembrane proteins and activate several signalling cascades, including the phospholipase c gamma , phosphatidylinositol- <dig> kinase  which activates akt/protein kinase b, and ras which activates extracellular signal-regulated protein kinase  pathways  <cit> .
fgf activity results in the control of gene expression through the modification of transcription factors by activated erks and akt.
as a consequence of altered gene expression, cellular proliferation, survival and fate determination can be governed by fgf activity.
how fgfs control gene expression and the nature of the genes that they regulate during development is still not completely established.
one step towards defining fgf target genes is to determine the temporal and spatial activity of fgfs during development.
this will provide an activity map of where and when these factors act to control developmental processes.
since activation of fgf signalling results in the phosphorylation of erk, one approach to illustrate fgf activity during development has been to detect the spatial and temporal presence of phosphorylated erks in the embryo.
this has resulted in mapping the location of fgf activity during mouse, chick, xenopus laevis and zebrafish embryogenesis  <cit> .
while these studies provide a detailed analysis of fgf activity during development, it is not possible to visualize fgf activity in the live embryo and observe the dynamic changes in fgf signalling as the embryo develops.
we have previously identified several fgf regulated genes in zebrafish, including the dual specificity phosphatase  <dig>  dusp <dig> , and sef, two genes that exhibit almost identical expression to fgf <dig> and fgf <dig> during development  <cit> .
dusp <dig> functions to dephosphorylate activated p <dig> and p <dig> erks and over-expression of dusp <dig> results in the suppression of fgf activity in the embryo  <cit> .
expression of dusp <dig> was suppressed in embryos treated with su <dig>  an fgfr inhibitor, or by the ectopic expression of dominant negative fgfr, indicating that dusp <dig> transcription is regulated by fgfs  <cit> .
genetic studies in mouse have identified the requirement for fgfrs in maintaining dusp <dig> expression, as loss of either fgfr <dig> or fgfr <dig> resulted in the depletion of dusp <dig> transcripts  <cit> .
it is clear from these studies that dusp <dig> expression is regulated by fgf ligands and receptors, however it has been controversial as to which signalling pathway downstream of the receptor is required for dusp <dig> gene transcription.
experiments described in the chick, mouse and zebrafish embryos have provided clues that dusp <dig> gene regulation is context dependent  <cit> .
in several studies, the pi3k inhibitor, ly <dig>  was used in the chick limb bud to show that blocking the pi3k/akt pathway results in the suppression of dusp <dig> expression within the distal limb bud  <cit> .
likewise, implantation beads soaked in ly <dig> could suppress dusp <dig> expression within the mid-hindbrain boundary  in mouse and chick embryos  <cit> .
in contrast, genetic studies in the mouse has revealed that the pi3k pathway is not required as knock-out of pdk <dig>  an upstream activator of pi3k, still allowed expression of dusp <dig> albeit in a disorganized fashion  <cit> .
analysis of the ras/mapk pathway under similar experimental conditions indicates that this pathway is also important for dusp <dig> gene expression.
for instance, the implantation of beads soaked with pd <dig>  a specific inhibitor of mek can also block dusp <dig> expression in the chick limb bud and somites, implicating the ras/mapk pathway in regulating dusp <dig> expression  <cit> .
further, over-expression of dusp <dig> itself, which would result in the removal of activated erks and hence the shutdown the ras/mapk pathway, resulted in the down regulation of dusp <dig> transcription  <cit> .
although these studies highlight the complex nature of dusp <dig> gene regulation downstream of the fgf receptor it is however clear that dusp <dig> is a direct target of fgf signalling.
this concept is supported by the fact that the presence of erk phosphorylation correlates with dusp <dig> expression throughout chick embryogenesis  <cit> .
a detailed map of erk activation was described and dusp <dig> expression was found to colocalize with activated erks in all tissues throughout chick development  <cit> .
thus fgf activity can be directly measured by either the presence of phosphorylated erks or indirectly by the presence of dusp <dig> transcripts.
the generation of transgenic lines that express fluorescent reporters in response to fgfs would allow live visualization of fgf activity during development.
since zebrafish embryos develop ex utero, the direct visualization of d2egfp in the embryos can provide an indirect biosensor for fgf activity in vivo.
in this report we have generated transgenic zebrafish lines that expresses a destabilized form of green fluorescent protein  under the control of active fgf signalling.
to achieve this we isolated the promoter region of the dusp <dig> gene and fused it to d2egfp reporter gene.
d2egfp fluorescence can be visualised in transgenic embryos within multiple tissues where activated erks  have been detected, and where expression of fgf ligands, receptors and target genes such as sef, sprouty <dig>  pea <dig> and erm have been described  <cit> .
the zebrafish has become a viable model organism for chemical screens  <cit> .
with the generation of fluorescent transgenic reporter zebrafish lines it is possible to discover specific molecules that can alter differentiation events during organogenesis  <cit> .
we have validated the fgf reporter lines as tools to identify novel fgf modulators and in counter screens it is possible to determine compound specificity towards two closely related rtk pathways.
the generation of in vivo reporters for fgf activity will provide a valuable tool to screen for genes or chemicals that modulate fgf signalling.
RESULTS
generation of transgenic fgf reporter lines
the zebrafish dusp <dig> gene locus was identified by pcr screening a bac library from genome systems.
the bac clone contained the full gene dusp <dig> locus, and a  <dig> kb fragment that includes the 5' untranslated sequence within exon i was subcloned into a vector containing the d2egfp, a gene that encodes a destabilised green fluorescent protein that has a two hour half-life   <cit> .
since fgf signalling during embryogenesis is dynamic, we reasoned that a destabilized form of egfp would likely recapitulate the endogenous expression of dusp <dig> mrna.
1-cell stage zebrafish embryos were co-injected with the transgenic dna construct and i-sce  <dig> meganuclease as described by thermes et al.
to generate transgenic lines  <cit> .
injected embryos were raised to adulthood and screened for potential germline carriers by d2egfp expression.
ten founder transgenic animals were identified in the f <dig> generation.
while d2egfp expression levels differed between each of the transgenic lines, the expression patterns were identical between the transgenic lines.
we maintained four of the strongest lines pt6-pt9), and for this study we utilized the line tgpt <dig>  we have generated homozygous lines for several of the transgenes and observed the expected mendelian inheritance.
further, there were no deleterious effects noted in transgenic animals, suggesting that insertion of the dna construct did not impact zebrafish growth and development.
expression of d2egfp is localized to fgf signalling centres in transgenic embryos
d2egfp was first visualized as early as dome stage within the prospective dorsal region of the embryo, consistent with the restricted dorsal expression of dusp <dig> mrna and with the initial expression patterns of fgf <dig> and - <dig>   <cit> .
the fluorescent pattern was supported by the expression of d2egfp mrna as tested by in situ hybridisation .
at sphere stage, d2egfp expression is maintained within the dorsal region and colocalises with dusp <dig> expression, a domain where the organiser  eventually forms .
at  <dig> hpf, the highest d2egfp expression is detected within the shield, and weakly throughout the margin, which resembles the pattern of dusp <dig> mrna at this stage .
reporter gene expression was also analysed at later stages by in situ hybridisation performed to detect the presence of d2egfp mrna.
at bud stage , d2egfp transcripts can be detected within the posterior ventral domain and within the presumptive hindbrain, where fgf <dig> expression has been noted   <cit> .
by  <dig> hpf, d2egfp mrna is detected within the mhb, pharyngeal arches, otic vesicle, retina, optic stalk and dorsal diencephalon .
the ligands fgf <dig>  fgf <dig> and fgf <dig>  and fgfrs are known to be expressed within these same domains, suggesting that mrna expression of this reporter gene is under the control of fgf signalling  <cit> .
in comparison to the fluorescent protein expression, d2egfp transcripts can be detected in a much wider domain and also more prominent .
one reason for the discrepancy can be attributed to the threshold level required for visualisation of d2egfp protein as compared to the detection of transcripts by in situ hybridisation.
at bud stage, fluorescent protein can be visualized within the medial neural plate, presumptive hindbrain and at a position that corresponds to the site where the dorsal forerunner cells  in the posterior domain of transgenic embryos have been described   <cit> .
the dfcs represent a specialized group of non-involuting cells that migrate just ahead of the shield during gastrulation and eventually forms a fluid filled structure known as kupffer's vesicle, a transient organ particular to teleosts  <cit> .
kupffer's vesicle is thought to serve equivalent functions in establishing left-right polarity as the mouse node  <cit> .
by the 6-somite stage, strong d2egfp fluorescence can be detected in the mhb, the hindbrain with strongest expression in r <dig>  the anterior lateral plate mesoderm and the kupffer's vesicle .
approximately two hours later at the 10-somite stage expression of d2egfp is located within the same domains as described for the 6-somite stage with the addition of the trigeminal ganglia .
the expression within kupffer's vesicle is quite striking as the structure is completely outlined by d2egfp positive cells from the 6-somite onwards .
in the zebrafish, a role for fgf signalling has been suggested in the formation of kupffer's vesicle, as fgf <dig> is expressed in the dfcs at gastrulation, and this structure is absent in about 30% of ace mutants  <cit> .
the tgpt <dig> line confirms that the dfcs and kupffer's vesicle receive fgf signals from the time when the dfcs begin to coalesce and right through to the 20-somite stage when kupffer's vesicle begins to collapse .
time lapse imaging of the transgenic embryos shows fluorescent dfcs migration towards the posterior region of the embryo and the formation of kupffer's vesicle at the 6-somite stage .
d2egfp expression is still noted throughout these stages and even after the collapse of kupffer's vescles when these epithelial cells migrate towards the tail bud and contribute to mesodermal tissues such as notochord, posterior somites and the tail bud   <cit> .
by the 26-somite stage, the kupffer's vesicle cells have lost their expression of d2egfp, suggesting that these cells no longer receive fgf signals .
at  <dig> hpf, d2egfp can be visualized within the dorsal retina, trigeminal ganglia, otic vesicles, within the dorsal diencephalon, and dorsal spinal cord neurons .
these domains of d2egfp expression are consistent with expression of fgf ligands, receptors and known target genes such as erm, pea <dig>  sef and sprouty <dig>  suggesting that the transgenic line reports on fgf activity in vivo  <cit> .
d2egfp expression persists throughout the next day of development in a majority of the same domains as at  <dig> hpf and continues up to  <dig> hpf .
the one exception is that dorsal retina expression is lost, while lens expression becomes stronger from  <dig> hpf onwards .
expression at later stages can also be detected within the developing jaw and further refined within the pharyngeal arches and the pectoral fins .
a dorsal view of a  <dig> hpf transgenic embryo reveals d2egfp expression within distinct cells in the dorsal diencephalon, suggesting that these particular cells are responding to fgf signals .
of particular interest is that there seems to be an asymmetric distribution of fluorescent cells with respect to the left-right axis, implying that within this region of the diencephalon, fgf signalling is asymmetrical .
an alternative view is that the asymmetric expression of d2egfp is a result of anatomical or stochastic  differences between the left and right side of the diencephalon.
studies have shown that the left habenular nuclei is larger and the parapineal gland is situated to the left of the midline, suggesting that d2egfp expression reflects structural difference between the left and right dorsal diencephalon  <cit> .
the domains of d2egfp expression in the transgenic embryos were consistent with many of the regions where endogenous dusp <dig> transcripts have been detected.
however, dusp <dig> mrna expression was not fully recapitulated in the transgenic embryos, in particular fgf activity and dusp <dig> expression have been well documented within the somites and tail bud and yet no expression of d2egfp mrna or protein were detected in these regions   <cit> .
one explanation is that only  <dig> kb of upstream dusp <dig> promoter sequence was used to generate the dna construct for transgenesis, hence it is likely that somitic and tail bud enhancers were not present within this sequence.
in summary we have generated several transgenic lines that express d2egfp under the control of the dusp <dig> promoter, a gene that is directly regulated by fgf signalling during development.
since d2egfp has a half life of just two hours, it is likely that domains of high d2egfp expression represents cells that respond to active fgf signalling as opposed to cells inheriting fluorescent protein from their ancestors.
fgf signalling controls reporter gene expression
to test whether d2egfp expression is responsive to experimental modulation in fgf signalling, we injected fgf <dig> mrna  into 2– <dig> cell stage tgpt <dig> embryos.
as expected, expression of d2egfp was greatly expanded in fgf8-injected embryos at the gastrula stage .
conversely, injection of fgf8-mos  resulted in a suppression of d2egfp expression within the mhb and retina, while d2egfp expression within the trigeminal ganglia was unaffected .
to confirm these findings we analyzed d2egfp expression in two mutant zebrafish lines that exhibit defective fgf signalling  <cit> .
we in-crossed tgpt <dig> into the ace mutant and expression of d2egfp within the mhb was greatly diminished in mutant embryos from  <dig> hpf onwards .
this is clearly evident at  <dig> hpf when the mhb is morphologically distinguishable between wildtype siblings and mutants .
furthermore expression within the retina and lens was absent, while expression in the optic stalk and trigeminal ganglia were unaffected at  <dig> hpf .
in the noi mutant, d2egfp expression was markedly reduced in the mhb, where fgf signalling is absent, however expression within the retina, lens, trigeminal ganglia and otic vesicles was unaltered.
in contrast to the ace mutants, expression of d2egfp within the optic stalk is severely reduced as this structure fails to from in the noi mutants  taken together, these results show that we have generated a transgenic reporter line that expresses d2egfp under the control of fgf signalling during development.
treatment of tgpt <dig> embryos with known fgf pathway inhibitors
given that tgpt <dig> embryos respond to changes in fgf signalling, these embryos can provide a valuable tool to screen for molecules that modulate fgf signalling in vivo.
to validate that these reporter lines can measure changes in fgf activity in a chemical screen, tgpt <dig> embryos were treated with known inhibitors of the fgf pathway.
for our studies we selected transgenic embryos at  <dig> hpf and treated with compounds for  <dig> hours.
we reasoned that since the half-life of d2egfp is  <dig> hours, then an inhibitor of fgf signalling should substantially reduce d2egfp fluorescence after 6– <dig> hours of treatment.
furthermore, since embryos at this developmental stage  have already formed many of the structures that express d2egfp such as the mhb, trigeminal ganglia and otic vesicles, then the assay would directly measure loss of d2egfp fluorescence as a consequence of fgf inhibition and not per se a loss of these tissues.
we treated transgenic embryos with the indolinone fgf receptor inhibitor, su <dig>   <cit> .
su <dig> has been extensively used to block fgf signalling in zebrafish and xenopus laevis embryos with doses ranging from  <dig> μm to  <dig> μm  <cit> .
in this study,  <dig> μm su <dig> was effective at suppressing expression of d2egfp within the trigeminal ganglia and reducing mhb fluorescence after only  <dig> hours of treatment .
at  <dig> μm of su <dig>  expression of d2egfp was markedly reduced within the mhb, and even eliminated within the dorsal retina and trigeminal ganglia .
at  <dig> μm, d2egfp fluorescence in the transgenic embryos was completely abolished .
treatment with other receptor tyrosine kinase inhibitors was also performed to determine the specificity of this assay for identifying other small molecules that can modulate fgf signalling.
we assessed the activity of several vascular endothelial growth factor receptor  and platelet derived growth factor receptor  inhibitors to reveal if these compounds, structurally related to su <dig>  could suppress d2egfp expression in the fgf reporter lines.
we reasoned that most of these compounds contain the same indolinone backbone as su <dig>  and therefore some would also block fgfr signalling in the zebrafish.
thus the analysis of these compounds in the fgf reporter line can determine the chemical structures that suppress fgf signalling from a family of related molecules.
one of the first indolinones to undergo clinical trials was su <dig>  a reported specific inhibitor for vegfr <dig>   <cit> .
su <dig> was added to  <dig> hpf stage transgenic embryos for  <dig> hours to determine if this compound is specific for vegfr versus fgfr.
while this compound exhibits autofluorescence, especially within the yolk, the expression of d2egfp did not markedly change at  <dig> μm .
in contrast at  <dig> μm, the trigeminal ganglia and mhb fluorescence was markedly diminished .
at even higher doses, su <dig> completely eliminated d2egfp expression within the transgenic embryos, even though the treated embryos exhibited strong fluorescent yolks, the expression of d2egfp within the mhb was clearly eliminated .
these results suggest that su <dig> is not completely specific to vegfr <dig> at these concentrations and it can block fgf signalling.
similarly, oxindole i was also effective at blocking d2egfp expression, however a much higher dose  was required , and .
su <dig> represents another indolinone inhibitor of vegfr, which has been shown to block autophosphorylation of vegfrs  <cit> .
in contrast to su <dig>  treatment up to  <dig> μm of su <dig> had little effect in the expression of d2egfp within the mhb and trigeminal ganglia in the tgpt <dig> embryos  and .
a newer generation of indolinone compound was also tested to determine its specificity towards vegfr/pdgfr versus fgfr.
su <dig>  has been shown to have at least 100-fold greater inhibitory effect targeting a vegfr and pdgfr versus fgfr   <cit> .
in our assays, su <dig> did not alter d2egfp expression suggesting that this compound does not block fgf signalling .
we also treated transgenic embryos with two structurally unrelated vegf signalling inhibitors, su <dig> and su <dig>  to determine the specificity of these molecules   <cit> .
both compounds have been shown to be effective at blocking vegfr, and to a lesser extent basic fgf in huvec tubulogenesis assays  <cit> .
treatment with high doses of su <dig> or su <dig> did not alter d2egfp fluorescence in the transgenic embryos, suggesting that these chemically divergent compounds do not block fgf signalling in the zebrafish at the doses indicated .
we next determined the activity of src kinase inhibitors and their role in fgf signalling in the early embryo as previous reports have shown that several src family members function to relay fgf signalling  <cit> .
pp <dig>  an inhibitor of several src kinases including fyn and lck as well as pdgfrs and bcr-abl, only mildly altered d2egfp expression at  <dig> μm , a dose that is several fold higher than what has been shown to inhibit fyn and lck in cell culture  <cit> .
su <dig>  a more specific inhibitor of src kinases, exhibited robust inhibitory activity in these assays.
at concentrations of  <dig> μm, expression of d2egfp was completely abolished within the mhb, trigeminal ganglia and the retina.
the concentration of su <dig> was within the range that is known to be specific for blocking mouse src kinase activity and not other related tyrosine kinases   <cit> .
in fact  <dig> μm su <dig> is likely to inhibit only the src members, yes, fyn, src and lyn, with yes kinase exhibiting higher sensitivity, suggesting that yes is required for fgf signalling in the zebrafish embryo  <cit> .
the results from these experiments support the use of transgenic fgf reporter lines to screen for small molecules that affect fgf signalling.
given the rapid nature of these screens as d2egfp expression can be suppressed in just  <dig> hours, it is likely that a chemical screen would identify compounds that directly modulate fgf signalling.
suppression of dusp <dig> expression in embryos treated with fgf inhibitors
to confirm that suppression of d2egfp fluorescence reflects reduced fgf target gene transcription, we analyzed dusp <dig> mrna expression in wildtype embryos treated with chemical inhibitors .
as predicted, compounds that suppressed fluorescence in tgpt6embryos also resulted in a reduction of dusp <dig> transcripts .
su <dig>  su <dig>  su <dig> and oxindole i all suppressed dusp <dig> expression after 10hr treatment at the dose indicated.
in particular, dusp <dig> expression within the dorsal diencephalon, pharyngeal endoderm and optic stalks were greatly reduced in embryos treated with su <dig>  s <dig>  su <dig> and oxindole i .
however, dusp <dig> expression in the mhb was either completely eliminated by su <dig> or only mildly affected by the other compounds in treated embryos .
these results suggest that the mhb represents a region of highest levels of fgf activity in the embryo during these developmental stages.
finally, compounds that did not alter overall d2egfp fluorescence  in treated fgf reporter line also did not exhibit changes in dusp <dig> transcript levels .
specificity of small molecule fgf signalling inhibitors can be evaluated with tgy <dig> transgenic embryos
since the results obtained with the vegfr inhibitors, su <dig>  su <dig> and su <dig>  were predominantly negative and did not alter d2egfp expression in the fgf reporter embryos, we wanted to confirm that these compounds were permeable in the embryo and have an effect on zebrafish vegr signalling.
one approach is to test the activity of these compounds in a zebrafish angiogenesis assay.
such assays have been developed to screen for novel antiangiogenic compounds that can block the formation of the zebrafish intersegmental vessels , and transgenic lines that delineate the isv have been used for such assays  <cit> .
for our studies we analysed isv outgrowth in the tgy <dig> line that expresses egfp under the control of the fli <dig> promoter  <cit> .
to determine the effects of these vegfr inhibitors on isv outgrowth, we treated tgy1transgenic embryos at  <dig> hpf for  <dig> hours.
treatment within this limited temporal window would reveal whether a compound could specifically block vegfr signalling and isv outgrowth as somites, dorsal aorta and posterior cardinal vein would have already been established at  <dig> hpf.
furthermore previous studies have highlighted the requirement for active vegfr signalling that is relayed through plcγ and akt to form the isv using a similar time frame  <cit> .
we first treated tgy <dig> embryos with su <dig>  su <dig> and su <dig>  compounds that did not alter fluorescence in the fgf reporter embryos, to determine if these compounds can block vegfr signalling in the zebrafish.
as predicted, all three compounds suppressed isv outgrowth in the transgenic embryos, thus exhibiting specificity towards vegfrs, and confirming that these compounds are permeable in the embryo .
similar effects on isv outgrowth were noted with oxindole i, a compound that blocked fgf signalling, however at a much lower dose  that was required to block fluorescence in tgpt <dig> embryos .
this result suggests that oxindole i has a higher activity towards vegfr signalling.
we next assayed the effects of the fgf receptor inhibitor, su <dig>  on isv outgrowth.
su <dig> treated tgy <dig> embryos also resulted in the suppression of isv formation .
the original description of su <dig> had provided evidence that this compound would interact with the atp binding domain of the vegfr and was supported by unpublished data that su <dig> did block vegfr signalling in tissue culture experiments, yet su <dig> is often used as a specific fgfr inhibitor.
thus we stress that the effects of su <dig> treatment may not exclusively reflect a blockade of fgfrs.
su <dig> and su <dig>  two compounds that have previously been shown to inhibit vegf signalling also inhibited isv outgrowth   <cit> .
thus su <dig> strongly suppressed both vegf and fgf signalling in our assays, while su <dig> was specific for vegf signalling.
in contrast, treatment of tgy <dig> embryos with the inhibitor of src kinases, su <dig>  did not block isv outgrowth, suggesting that vegf signalling does not require src kinases to direct vessel formation .
to confirm our findings that vegfr inhibitors did indeed block intersegmental vessel outgrowth, we performed in situ hybridisation studies to detect the presence of these vessels through fli <dig> expression.
treatment of embryos with  <dig> μm oxindole  <dig>   <dig> μm su <dig>   <dig> μm su <dig>   <dig> μm su <dig>  or with  <dig> μm su <dig> prevented isv formation as determined by loss of fli <dig> expression in the intersegmental sprouts .
again these results confirm that su <dig> can block vegfr signalling in the zebrafish and is not a specific inhibitor of fgfrs.
in contrast, expression of fli <dig> within the isv was unaffected in embryos treated with  <dig> μm su <dig>  confirming the notion that src kinases are not required in isv outgrowth .
discussion
in this paper we present the generation of a zebrafish transgenic line that reports on fgf signalling during embryonic development.
expression of d2egfp in these embryos is regulated by fgf signalling and maps to domains where activated erks have been detected  <cit> .
time-lapse imaging of transgenic embryos shows the dynamic expression of d2egfp throughout embryogenesis.
these movies demarcate the formation of kupffer's vesicle, as d2egfp labels the dfcs from gastrulation onwards.
since kupffer's vesicle plays a critical role in the determination of left-right polarity these lines will provide a valuable tool to access the integrity of kupffer's vesicle formation in left-right polarity mutants  <cit> .
furthermore, these lines provides a way to identify dfcs in post-gastrulation but pre-kupffer's vesicle stages in live embryos.
to our knowledge this is the only line that specifically labels the dfcs with fluorescence as they migrate during gastrulation to eventually form kupffer's vesicle.
this is in contrast to the tg line that also expresses gfp in kupffer's vesicle, as gfp expression was only detected once the vesicle had formed at the 6-somite stage  <cit> .
while a majority of the d2egfp expression resembles that of dusp <dig> mrna during development, a notable exception is the lack of d2egfp expression within the developing somites and tail bud, two domains where fgfs and erk activation have been described  <cit> .
since only  <dig> kb of upstream promoter sequence was used in generating the reporter construct, it is likely that the somitic and tail bud enhancers elements are not part of this sequence.
the expression of d2egfp is under the control of fgf signalling as manipulation of fgf activity in transgenic embryos resulted in altered d2egfp expression.
these lines will provide useful tools to analyze the integrity of fgf signalling under experimental conditions such as antisense mos injections or in mutant strains.
previously, balciunas et al.
described a transposon-mediated enhancer trapped line that contains an egfp reporter inserted  <dig> kb within the dusp <dig> locus   <cit> .
in the et <dig> transgenic embryos, egfp is detected within the mhb, pharyngeal endoderm and somites at  <dig> hpf, suggesting that dusp <dig> enhancers may regulate the expression of egfp  <cit> .
however, it has not been demonstrated if egfp expression in the et <dig> embryos can respond to experimental changes in fgf signalling  <cit> .
we believe that tgpt <dig> represent the first description of vertebrate transgenic embryos that can report on fgf activity in vivo.
we have used these transgenic lines to validate an approach to screen for small molecules that can modulate fgf signalling.
our results show that within  <dig> hours of treatment with the fgfr inhibitor, su <dig>  complete suppression of de2gfp expression ensued, confirming that these lines can rapidly identify compounds that modulate fgf signal transduction in the embryo.
since fgfrs are members of a larger family of receptor tyrosine kinases we wanted to determine if compounds that are structurally related to su <dig> could also block fgf signalling in the embryo.
we determined the specificity of several vegfr and pdgfr inhibitors that contain the same indolinone backbone as su <dig>  from our studies it was clear that su <dig> and oxindole i, two inhibitors of vegfr could block d2egfp expression in the fgf reporter embryos.
furthermore, from these studies we also revealed a putative role for src kinases in relaying fgf signals in the zebrafish as treatment with src kinase inhibitors, pp <dig> and su <dig>  suppressed d2egfp fluorescence.
since several members of this kinase family are expressed early and within cells that receive fgf signals in the developing embryo, including yes and fyn, the preferred targets of su <dig>  it is reasonable to think that these kinases are involved in fgf signalling  <cit> .
we have coupled the pilot screen with the tgy <dig> transgenic line to assay the specificity of these compounds between fgfr and vegfr signalling.
we confirm that su <dig>  su <dig>  su <dig>  oxindole i and surprisingly su <dig> exhibited inhibitory effects on isv outgrowth and probably a result of vegfr inhibition.
in the original study, a crystal structure of su <dig> complexed with the fgfr <dig> kinase domain highlighted the exact peptide residues that interfaced with the compound  <cit> .
since these residues are also highly conserved with the vegfrs kinase domain, it was postulated that su <dig> could interact with the vegfrs  <cit> .
furthermore the authors refer to unpublished observations that su <dig> did inhibit vegfr signalling in living cells  <cit> .
our studies reveal that su <dig> can block isv outgrowth, which we interpret as a result of vegfr inhibition in the zebrafish embryo.
alternatively, our data could imply that fgfrs are required for isv outgrowths given that fgfs are known to play a role in angiogenesis  <cit> .
however from the published literature, fgfr1- <dig> are not expressed in the isv to support the role for this pathway in vasculogenesis  <cit> .
su <dig> has been used extensively as a fgfr inhibitor in many studies.
in light of these findings, other experiments should be considered to test whether su <dig> might also block vegfr signalling to elicit the observed phenotypes.
CONCLUSIONS
this study describes the generation of transgenic zebrafish, tgpt <dig>  that reports on fgf activity during development.
the expression of d2egfp mirrors the expression of several fgf ligands in the early embryo and will provide a tool to analyse fgf signalling under various experimental conditions.
we have performed a pilot screen to validate these lines in chemical screens to identify novel compounds that can modulate fgf activity.
this rapid screening protocol can be coupled with the tgy <dig> line to eliminate compounds that can potentially cross react with the vegfr pathway.
finally, acridine orange staining will further eliminate toxic compounds, thus by following these procedures it is possible to identify chemicals that specifically modulate fgf signalling in vivo.

 the complex process of embryogenesis is directed by the regulation of signalling pathways that are achieved in part by the activity of a variety of secreted ligands. thus understanding the temporal and spatial activity of signalling peptides is key to determining the role for these factors in controlling cellular fates. for example, fibroblast growth factors, a family of secreted glycoproteins, perform crucial functions that include the establishment of embryo polarity, the formation of organizing centres, and the induction of limb outgrowth. these ligands are expressed in discrete domains during development and their actions are restricted to cells that express integral membrane proteins that can bind fgfs. the fgf receptors are members of the receptor tyrosine kinase class of transmembrane proteins and activate several signalling cascades, including the phospholipase c gamma, phosphatidylinositol- kinase which activates akt/protein kinase b, and ras which activates extracellular signal-regulated protein kinase pathways. fgf activity results in the control of gene expression through the modification of transcription factors by activated erks and akt. as a consequence of altered gene expression, cellular proliferation, survival and fate determination can be governed by fgf activity. how fgfs control gene expression and the nature of the genes that they regulate during development is still not completely established. one step towards defining fgf target genes is to determine the temporal and spatial activity of fgfs during development. this will provide an activity map of where and when these factors act to control developmental processes. since activation of fgf signalling results in the phosphorylation of erk, one approach to illustrate fgf activity during development has been to detect the spatial and temporal presence of phosphorylated erks in the embryo. this has resulted in mapping the location of fgf activity during mouse, chick, xenopus laevis and zebrafish embryogenesis. while these studies provide a detailed analysis of fgf activity during development, it is not possible to visualize fgf activity in the live embryo and observe the dynamic changes in fgf signalling as the embryo develops. we have previously identified several fgf regulated genes in zebrafish, including the dual specificity phosphatase dusp, and sef, two genes that exhibit almost identical expression to fgf and fgf during development. dusp functions to dephosphorylate activated p and p erks and over-expression of dusp results in the suppression of fgf activity in the embryo. expression of dusp was suppressed in embryos treated with su an fgfr inhibitor, or by the ectopic expression of dominant negative fgfr, indicating that dusp transcription is regulated by fgfs. genetic studies in mouse have identified the requirement for fgfrs in maintaining dusp expression, as loss of either fgfr or fgfr resulted in the depletion of dusp transcripts. it is clear from these studies that dusp expression is regulated by fgf ligands and receptors, however it has been controversial as to which signalling pathway downstream of the receptor is required for dusp gene transcription. experiments described in the chick, mouse and zebrafish embryos have provided clues that dusp gene regulation is context dependent. in several studies, the pi3k inhibitor, ly was used in the chick limb bud to show that blocking the pi3k/akt pathway results in the suppression of dusp expression within the distal limb bud. likewise, implantation beads soaked in ly could suppress dusp expression within the mid-hindbrain boundary in mouse and chick embryos. in contrast, genetic studies in the mouse has revealed that the pi3k pathway is not required as knock-out of pdk an upstream activator of pi3k, still allowed expression of dusp albeit in a disorganized fashion. analysis of the ras/mapk pathway under similar experimental conditions indicates that this pathway is also important for dusp gene expression. for instance, the implantation of beads soaked with pd a specific inhibitor of mek can also block dusp expression in the chick limb bud and somites, implicating the ras/mapk pathway in regulating dusp expression. further, over-expression of dusp itself, which would result in the removal of activated erks and hence the shutdown the ras/mapk pathway, resulted in the down regulation of dusp transcription. although these studies highlight the complex nature of dusp gene regulation downstream of the fgf receptor it is however clear that dusp is a direct target of fgf signalling. this concept is supported by the fact that the presence of erk phosphorylation correlates with dusp expression throughout chick embryogenesis. a detailed map of erk activation was described and dusp expression was found to colocalize with activated erks in all tissues throughout chick development. thus fgf activity can be directly measured by either the presence of phosphorylated erks or indirectly by the presence of dusp transcripts. the generation of transgenic lines that express fluorescent reporters in response to fgfs would allow live visualization of fgf activity during development. since zebrafish embryos develop ex utero, the direct visualization of d2egfp in the embryos can provide an indirect biosensor for fgf activity in vivo. in this report we have generated transgenic zebrafish lines that expresses a destabilized form of green fluorescent protein under the control of active fgf signalling. to achieve this we isolated the promoter region of the dusp gene and fused it to d2egfp reporter gene. d2egfp fluorescence can be visualised in transgenic embryos within multiple tissues where activated erks have been detected, and where expression of fgf ligands, receptors and target genes such as sef, sprouty pea and erm have been described. the zebrafish has become a viable model organism for chemical screens. with the generation of fluorescent transgenic reporter zebrafish lines it is possible to discover specific molecules that can alter differentiation events during organogenesis. we have validated the fgf reporter lines as tools to identify novel fgf modulators and in counter screens it is possible to determine compound specificity towards two closely related rtk pathways. the generation of in vivo reporters for fgf activity will provide a valuable tool to screen for genes or chemicals that modulate fgf signalling.  generation of transgenic fgf reporter lines the zebrafish dusp gene locus was identified by pcr screening a bac library from genome systems. the bac clone contained the full gene dusp locus, and a kb fragment that includes the 5' untranslated sequence within exon i was subcloned into a vector containing the d2egfp, a gene that encodes a destabilised green fluorescent protein that has a two hour half-life. since fgf signalling during embryogenesis is dynamic, we reasoned that a destabilized form of egfp would likely recapitulate the endogenous expression of dusp mrna. 1-cell stage zebrafish embryos were co-injected with the transgenic dna construct and i-sce meganuclease as described by thermes et al. to generate transgenic lines. injected embryos were raised to adulthood and screened for potential germline carriers by d2egfp expression. ten founder transgenic animals were identified in the f generation. while d2egfp expression levels differed between each of the transgenic lines, the expression patterns were identical between the transgenic lines. we maintained four of the strongest lines pt6-pt9), and for this study we utilized the line tgpt we have generated homozygous lines for several of the transgenes and observed the expected mendelian inheritance. further, there were no deleterious effects noted in transgenic animals, suggesting that insertion of the dna construct did not impact zebrafish growth and development. expression of d2egfp is localized to fgf signalling centres in transgenic embryos d2egfp was first visualized as early as dome stage within the prospective dorsal region of the embryo, consistent with the restricted dorsal expression of dusp mrna and with the initial expression patterns of fgf and -. the fluorescent pattern was supported by the expression of d2egfp mrna as tested by in situ hybridisation. at sphere stage, d2egfp expression is maintained within the dorsal region and colocalises with dusp expression, a domain where the organiser eventually forms. at hpf, the highest d2egfp expression is detected within the shield, and weakly throughout the margin, which resembles the pattern of dusp mrna at this stage. reporter gene expression was also analysed at later stages by in situ hybridisation performed to detect the presence of d2egfp mrna. at bud stage, d2egfp transcripts can be detected within the posterior ventral domain and within the presumptive hindbrain, where fgf expression has been noted. by hpf, d2egfp mrna is detected within the mhb, pharyngeal arches, otic vesicle, retina, optic stalk and dorsal diencephalon. the ligands fgf fgf and fgf and fgfrs are known to be expressed within these same domains, suggesting that mrna expression of this reporter gene is under the control of fgf signalling. in comparison to the fluorescent protein expression, d2egfp transcripts can be detected in a much wider domain and also more prominent. one reason for the discrepancy can be attributed to the threshold level required for visualisation of d2egfp protein as compared to the detection of transcripts by in situ hybridisation. at bud stage, fluorescent protein can be visualized within the medial neural plate, presumptive hindbrain and at a position that corresponds to the site where the dorsal forerunner cells in the posterior domain of transgenic embryos have been described. the dfcs represent a specialized group of non-involuting cells that migrate just ahead of the shield during gastrulation and eventually forms a fluid filled structure known as kupffer's vesicle, a transient organ particular to teleosts. kupffer's vesicle is thought to serve equivalent functions in establishing left-right polarity as the mouse node. by the 6-somite stage, strong d2egfp fluorescence can be detected in the mhb, the hindbrain with strongest expression in r the anterior lateral plate mesoderm and the kupffer's vesicle. approximately two hours later at the 10-somite stage expression of d2egfp is located within the same domains as described for the 6-somite stage with the addition of the trigeminal ganglia. the expression within kupffer's vesicle is quite striking as the structure is completely outlined by d2egfp positive cells from the 6-somite onwards. in the zebrafish, a role for fgf signalling has been suggested in the formation of kupffer's vesicle, as fgf is expressed in the dfcs at gastrulation, and this structure is absent in about 30% of ace mutants. the tgpt line confirms that the dfcs and kupffer's vesicle receive fgf signals from the time when the dfcs begin to coalesce and right through to the 20-somite stage when kupffer's vesicle begins to collapse. time lapse imaging of the transgenic embryos shows fluorescent dfcs migration towards the posterior region of the embryo and the formation of kupffer's vesicle at the 6-somite stage. d2egfp expression is still noted throughout these stages and even after the collapse of kupffer's vescles when these epithelial cells migrate towards the tail bud and contribute to mesodermal tissues such as notochord, posterior somites and the tail bud. by the 26-somite stage, the kupffer's vesicle cells have lost their expression of d2egfp, suggesting that these cells no longer receive fgf signals. at hpf, d2egfp can be visualized within the dorsal retina, trigeminal ganglia, otic vesicles, within the dorsal diencephalon, and dorsal spinal cord neurons. these domains of d2egfp expression are consistent with expression of fgf ligands, receptors and known target genes such as erm, pea sef and sprouty suggesting that the transgenic line reports on fgf activity in vivo. d2egfp expression persists throughout the next day of development in a majority of the same domains as at hpf and continues up to hpf. the one exception is that dorsal retina expression is lost, while lens expression becomes stronger from hpf onwards. expression at later stages can also be detected within the developing jaw and further refined within the pharyngeal arches and the pectoral fins. a dorsal view of a hpf transgenic embryo reveals d2egfp expression within distinct cells in the dorsal diencephalon, suggesting that these particular cells are responding to fgf signals. of particular interest is that there seems to be an asymmetric distribution of fluorescent cells with respect to the left-right axis, implying that within this region of the diencephalon, fgf signalling is asymmetrical. an alternative view is that the asymmetric expression of d2egfp is a result of anatomical or stochastic differences between the left and right side of the diencephalon. studies have shown that the left habenular nuclei is larger and the parapineal gland is situated to the left of the midline, suggesting that d2egfp expression reflects structural difference between the left and right dorsal diencephalon. the domains of d2egfp expression in the transgenic embryos were consistent with many of the regions where endogenous dusp transcripts have been detected. however, dusp mrna expression was not fully recapitulated in the transgenic embryos, in particular fgf activity and dusp expression have been well documented within the somites and tail bud and yet no expression of d2egfp mrna or protein were detected in these regions. one explanation is that only kb of upstream dusp promoter sequence was used to generate the dna construct for transgenesis, hence it is likely that somitic and tail bud enhancers were not present within this sequence. in summary we have generated several transgenic lines that express d2egfp under the control of the dusp promoter, a gene that is directly regulated by fgf signalling during development. since d2egfp has a half life of just two hours, it is likely that domains of high d2egfp expression represents cells that respond to active fgf signalling as opposed to cells inheriting fluorescent protein from their ancestors. fgf signalling controls reporter gene expression to test whether d2egfp expression is responsive to experimental modulation in fgf signalling, we injected fgf mrna into 2– cell stage tgpt embryos. as expected, expression of d2egfp was greatly expanded in fgf8-injected embryos at the gastrula stage. conversely, injection of fgf8-mos resulted in a suppression of d2egfp expression within the mhb and retina, while d2egfp expression within the trigeminal ganglia was unaffected. to confirm these findings we analyzed d2egfp expression in two mutant zebrafish lines that exhibit defective fgf signalling. we in-crossed tgpt into the ace mutant and expression of d2egfp within the mhb was greatly diminished in mutant embryos from hpf onwards. this is clearly evident at hpf when the mhb is morphologically distinguishable between wildtype siblings and mutants. furthermore expression within the retina and lens was absent, while expression in the optic stalk and trigeminal ganglia were unaffected at hpf. in the noi mutant, d2egfp expression was markedly reduced in the mhb, where fgf signalling is absent, however expression within the retina, lens, trigeminal ganglia and otic vesicles was unaltered. in contrast to the ace mutants, expression of d2egfp within the optic stalk is severely reduced as this structure fails to from in the noi mutants taken together, these results show that we have generated a transgenic reporter line that expresses d2egfp under the control of fgf signalling during development. treatment of tgpt embryos with known fgf pathway inhibitors given that tgpt embryos respond to changes in fgf signalling, these embryos can provide a valuable tool to screen for molecules that modulate fgf signalling in vivo. to validate that these reporter lines can measure changes in fgf activity in a chemical screen, tgpt embryos were treated with known inhibitors of the fgf pathway. for our studies we selected transgenic embryos at hpf and treated with compounds for hours. we reasoned that since the half-life of d2egfp is hours, then an inhibitor of fgf signalling should substantially reduce d2egfp fluorescence after 6– hours of treatment. furthermore, since embryos at this developmental stage have already formed many of the structures that express d2egfp such as the mhb, trigeminal ganglia and otic vesicles, then the assay would directly measure loss of d2egfp fluorescence as a consequence of fgf inhibition and not per se a loss of these tissues. we treated transgenic embryos with the indolinone fgf receptor inhibitor, su. su has been extensively used to block fgf signalling in zebrafish and xenopus laevis embryos with doses ranging from μm to μm. in this study, μm su was effective at suppressing expression of d2egfp within the trigeminal ganglia and reducing mhb fluorescence after only hours of treatment. at μm of su expression of d2egfp was markedly reduced within the mhb, and even eliminated within the dorsal retina and trigeminal ganglia. at μm, d2egfp fluorescence in the transgenic embryos was completely abolished. treatment with other receptor tyrosine kinase inhibitors was also performed to determine the specificity of this assay for identifying other small molecules that can modulate fgf signalling. we assessed the activity of several vascular endothelial growth factor receptor and platelet derived growth factor receptor inhibitors to reveal if these compounds, structurally related to su could suppress d2egfp expression in the fgf reporter lines. we reasoned that most of these compounds contain the same indolinone backbone as su and therefore some would also block fgfr signalling in the zebrafish. thus the analysis of these compounds in the fgf reporter line can determine the chemical structures that suppress fgf signalling from a family of related molecules. one of the first indolinones to undergo clinical trials was su a reported specific inhibitor for vegfr. su was added to hpf stage transgenic embryos for hours to determine if this compound is specific for vegfr versus fgfr. while this compound exhibits autofluorescence, especially within the yolk, the expression of d2egfp did not markedly change at μm. in contrast at μm, the trigeminal ganglia and mhb fluorescence was markedly diminished. at even higher doses, su completely eliminated d2egfp expression within the transgenic embryos, even though the treated embryos exhibited strong fluorescent yolks, the expression of d2egfp within the mhb was clearly eliminated. these results suggest that su is not completely specific to vegfr at these concentrations and it can block fgf signalling. similarly, oxindole i was also effective at blocking d2egfp expression, however a much higher dose was required, and. su represents another indolinone inhibitor of vegfr, which has been shown to block autophosphorylation of vegfrs. in contrast to su treatment up to μm of su had little effect in the expression of d2egfp within the mhb and trigeminal ganglia in the tgpt embryos and. a newer generation of indolinone compound was also tested to determine its specificity towards vegfr/pdgfr versus fgfr. su has been shown to have at least 100-fold greater inhibitory effect targeting a vegfr and pdgfr versus fgfr. in our assays, su did not alter d2egfp expression suggesting that this compound does not block fgf signalling. we also treated transgenic embryos with two structurally unrelated vegf signalling inhibitors, su and su to determine the specificity of these molecules. both compounds have been shown to be effective at blocking vegfr, and to a lesser extent basic fgf in huvec tubulogenesis assays. treatment with high doses of su or su did not alter d2egfp fluorescence in the transgenic embryos, suggesting that these chemically divergent compounds do not block fgf signalling in the zebrafish at the doses indicated. we next determined the activity of src kinase inhibitors and their role in fgf signalling in the early embryo as previous reports have shown that several src family members function to relay fgf signalling. pp an inhibitor of several src kinases including fyn and lck as well as pdgfrs and bcr-abl, only mildly altered d2egfp expression at μm, a dose that is several fold higher than what has been shown to inhibit fyn and lck in cell culture. su a more specific inhibitor of src kinases, exhibited robust inhibitory activity in these assays. at concentrations of μm, expression of d2egfp was completely abolished within the mhb, trigeminal ganglia and the retina. the concentration of su was within the range that is known to be specific for blocking mouse src kinase activity and not other related tyrosine kinases. in fact μm su is likely to inhibit only the src members, yes, fyn, src and lyn, with yes kinase exhibiting higher sensitivity, suggesting that yes is required for fgf signalling in the zebrafish embryo. the results from these experiments support the use of transgenic fgf reporter lines to screen for small molecules that affect fgf signalling. given the rapid nature of these screens as d2egfp expression can be suppressed in just hours, it is likely that a chemical screen would identify compounds that directly modulate fgf signalling. suppression of dusp expression in embryos treated with fgf inhibitors to confirm that suppression of d2egfp fluorescence reflects reduced fgf target gene transcription, we analyzed dusp mrna expression in wildtype embryos treated with chemical inhibitors. as predicted, compounds that suppressed fluorescence in tgpt6embryos also resulted in a reduction of dusp transcripts. su su su and oxindole i all suppressed dusp expression after 10hr treatment at the dose indicated. in particular, dusp expression within the dorsal diencephalon, pharyngeal endoderm and optic stalks were greatly reduced in embryos treated with su s su and oxindole i. however, dusp expression in the mhb was either completely eliminated by su or only mildly affected by the other compounds in treated embryos. these results suggest that the mhb represents a region of highest levels of fgf activity in the embryo during these developmental stages. finally, compounds that did not alter overall d2egfp fluorescence in treated fgf reporter line also did not exhibit changes in dusp transcript levels. specificity of small molecule fgf signalling inhibitors can be evaluated with tgy transgenic embryos since the results obtained with the vegfr inhibitors, su su and su were predominantly negative and did not alter d2egfp expression in the fgf reporter embryos, we wanted to confirm that these compounds were permeable in the embryo and have an effect on zebrafish vegr signalling. one approach is to test the activity of these compounds in a zebrafish angiogenesis assay. such assays have been developed to screen for novel antiangiogenic compounds that can block the formation of the zebrafish intersegmental vessels, and transgenic lines that delineate the isv have been used for such assays. for our studies we analysed isv outgrowth in the tgy line that expresses egfp under the control of the fli promoter. to determine the effects of these vegfr inhibitors on isv outgrowth, we treated tgy1transgenic embryos at hpf for hours. treatment within this limited temporal window would reveal whether a compound could specifically block vegfr signalling and isv outgrowth as somites, dorsal aorta and posterior cardinal vein would have already been established at hpf. furthermore previous studies have highlighted the requirement for active vegfr signalling that is relayed through plcγ and akt to form the isv using a similar time frame. we first treated tgy embryos with su su and su compounds that did not alter fluorescence in the fgf reporter embryos, to determine if these compounds can block vegfr signalling in the zebrafish. as predicted, all three compounds suppressed isv outgrowth in the transgenic embryos, thus exhibiting specificity towards vegfrs, and confirming that these compounds are permeable in the embryo. similar effects on isv outgrowth were noted with oxindole i, a compound that blocked fgf signalling, however at a much lower dose that was required to block fluorescence in tgpt embryos. this result suggests that oxindole i has a higher activity towards vegfr signalling. we next assayed the effects of the fgf receptor inhibitor, su on isv outgrowth. su treated tgy embryos also resulted in the suppression of isv formation. the original description of su had provided evidence that this compound would interact with the atp binding domain of the vegfr and was supported by unpublished data that su did block vegfr signalling in tissue culture experiments, yet su is often used as a specific fgfr inhibitor. thus we stress that the effects of su treatment may not exclusively reflect a blockade of fgfrs. su and su two compounds that have previously been shown to inhibit vegf signalling also inhibited isv outgrowth. thus su strongly suppressed both vegf and fgf signalling in our assays, while su was specific for vegf signalling. in contrast, treatment of tgy embryos with the inhibitor of src kinases, su did not block isv outgrowth, suggesting that vegf signalling does not require src kinases to direct vessel formation. to confirm our findings that vegfr inhibitors did indeed block intersegmental vessel outgrowth, we performed in situ hybridisation studies to detect the presence of these vessels through fli expression. treatment of embryos with μm oxindole μm su μm su μm su or with μm su prevented isv formation as determined by loss of fli expression in the intersegmental sprouts. again these results confirm that su can block vegfr signalling in the zebrafish and is not a specific inhibitor of fgfrs. in contrast, expression of fli within the isv was unaffected in embryos treated with μm su confirming the notion that src kinases are not required in isv outgrowth. discussion in this paper we present the generation of a zebrafish transgenic line that reports on fgf signalling during embryonic development. expression of d2egfp in these embryos is regulated by fgf signalling and maps to domains where activated erks have been detected. time-lapse imaging of transgenic embryos shows the dynamic expression of d2egfp throughout embryogenesis. these movies demarcate the formation of kupffer's vesicle, as d2egfp labels the dfcs from gastrulation onwards. since kupffer's vesicle plays a critical role in the determination of left-right polarity these lines will provide a valuable tool to access the integrity of kupffer's vesicle formation in left-right polarity mutants. furthermore, these lines provides a way to identify dfcs in post-gastrulation but pre-kupffer's vesicle stages in live embryos. to our knowledge this is the only line that specifically labels the dfcs with fluorescence as they migrate during gastrulation to eventually form kupffer's vesicle. this is in contrast to the tg line that also expresses gfp in kupffer's vesicle, as gfp expression was only detected once the vesicle had formed at the 6-somite stage. while a majority of the d2egfp expression resembles that of dusp mrna during development, a notable exception is the lack of d2egfp expression within the developing somites and tail bud, two domains where fgfs and erk activation have been described. since only kb of upstream promoter sequence was used in generating the reporter construct, it is likely that the somitic and tail bud enhancers elements are not part of this sequence. the expression of d2egfp is under the control of fgf signalling as manipulation of fgf activity in transgenic embryos resulted in altered d2egfp expression. these lines will provide useful tools to analyze the integrity of fgf signalling under experimental conditions such as antisense mos injections or in mutant strains. previously, balciunas et al. described a transposon-mediated enhancer trapped line that contains an egfp reporter inserted kb within the dusp locus. in the et transgenic embryos, egfp is detected within the mhb, pharyngeal endoderm and somites at hpf, suggesting that dusp enhancers may regulate the expression of egfp. however, it has not been demonstrated if egfp expression in the et embryos can respond to experimental changes in fgf signalling. we believe that tgpt represent the first description of vertebrate transgenic embryos that can report on fgf activity in vivo. we have used these transgenic lines to validate an approach to screen for small molecules that can modulate fgf signalling. our results show that within hours of treatment with the fgfr inhibitor, su complete suppression of de2gfp expression ensued, confirming that these lines can rapidly identify compounds that modulate fgf signal transduction in the embryo. since fgfrs are members of a larger family of receptor tyrosine kinases we wanted to determine if compounds that are structurally related to su could also block fgf signalling in the embryo. we determined the specificity of several vegfr and pdgfr inhibitors that contain the same indolinone backbone as su from our studies it was clear that su and oxindole i, two inhibitors of vegfr could block d2egfp expression in the fgf reporter embryos. furthermore, from these studies we also revealed a putative role for src kinases in relaying fgf signals in the zebrafish as treatment with src kinase inhibitors, pp and su suppressed d2egfp fluorescence. since several members of this kinase family are expressed early and within cells that receive fgf signals in the developing embryo, including yes and fyn, the preferred targets of su it is reasonable to think that these kinases are involved in fgf signalling. we have coupled the pilot screen with the tgy transgenic line to assay the specificity of these compounds between fgfr and vegfr signalling. we confirm that su su su oxindole i and surprisingly su exhibited inhibitory effects on isv outgrowth and probably a result of vegfr inhibition. in the original study, a crystal structure of su complexed with the fgfr kinase domain highlighted the exact peptide residues that interfaced with the compound. since these residues are also highly conserved with the vegfrs kinase domain, it was postulated that su could interact with the vegfrs. furthermore the authors refer to unpublished observations that su did inhibit vegfr signalling in living cells. our studies reveal that su can block isv outgrowth, which we interpret as a result of vegfr inhibition in the zebrafish embryo. alternatively, our data could imply that fgfrs are required for isv outgrowths given that fgfs are known to play a role in angiogenesis. however from the published literature, fgfr1- are not expressed in the isv to support the role for this pathway in vasculogenesis. su has been used extensively as a fgfr inhibitor in many studies. in light of these findings, other experiments should be considered to test whether su might also block vegfr signalling to elicit the observed phenotypes.  this study describes the generation of transgenic zebrafish, tgpt that reports on fgf activity during development. the expression of d2egfp mirrors the expression of several fgf ligands in the early embryo and will provide a tool to analyse fgf signalling under various experimental conditions. we have performed a pilot screen to validate these lines in chemical screens to identify novel compounds that can modulate fgf activity. this rapid screening protocol can be coupled with the tgy line to eliminate compounds that can potentially cross react with the vegfr pathway. finally, acridine orange staining will further eliminate toxic compounds, thus by following these procedures it is possible to identify chemicals that specifically modulate fgf signalling in vivo. 
this includes the mid-hindbrain boundary, pharyngeal endoderm, otic vesicle, hindbrain, and kupffer's vesicle. these compounds were counter screened with the transgenic line tgy that serves as an indirect read-out for vascular endothelial growth factor signalling in order to determine the specificity between related receptor tyrosine kinases. the dusp promoter was isolated from zebrafish and used to drive expression of destabilized green fluorescent protein in transgenic embryos ). the expression of d2egfp is under the control of fgf signalling as treatment with fgf receptor inhibitors results in the suppression of d2egfp expression. at later stages, d2egfp is detected within structures that correlate with the expression of fgf ligands and their receptors. the generation of transgenic reporter zebrafish lines has allowed direct visualization of fgf signalling within the developing embryo. in a pilot screen of commercially available small molecules we have evaluated the effectiveness of the transgenic lines to identify specific fgf inhibitors within the class of indolinones. these fgf reporter transgenic lines provide a tool to screen for specific compounds that can distinguish between two conserved members of the rtk family. fibroblast growth factors represent a large family of secreted proteins that are required for proper development and physiological processes.
 fibroblast growth factors represent a large family of secreted proteins that are required for proper development and physiological processes. mutations in mouse and zebrafish fgfs result in abnormal embryogenesis and lethality. a key to understanding the precise role for these factors is to determine their spatial and temporal activity during embryogenesis.  expression of dual specificity phosphatase is controlled by fgf signalling throughout development. the dusp promoter was isolated from zebrafish and used to drive expression of destabilized green fluorescent protein in transgenic embryos ). expression of d2egfp is initiated as early as hours post-fertilization within the future dorsal region of the embryo, where fgf and fgf are initially expressed. at later stages, d2egfp is detected within structures that correlate with the expression of fgf ligands and their receptors. this includes the mid-hindbrain boundary, pharyngeal endoderm, otic vesicle, hindbrain, and kupffer's vesicle. the expression of d2egfp is under the control of fgf signalling as treatment with fgf receptor inhibitors results in the suppression of d2egfp expression. in a pilot screen of commercially available small molecules we have evaluated the effectiveness of the transgenic lines to identify specific fgf inhibitors within the class of indolinones. these compounds were counter screened with the transgenic line tgy that serves as an indirect read-out for vascular endothelial growth factor signalling in order to determine the specificity between related receptor tyrosine kinases. from these assays it is possible to determine the specificity of these indolinones towards specific rtk signalling pathways. this has enabled the identification of compounds that can block specifically the vegfr or the fgfr signalling pathway.  the generation of transgenic reporter zebrafish lines has allowed direct visualization of fgf signalling within the developing embryo. these fgf reporter transgenic lines provide a tool to screen for specific compounds that can distinguish between two conserved members of the rtk family. 