BACKGROUND
vertebrate gastrulation is a highly coordinated process that leads to formation of the three primary germ layers  and sets up the body plan for subsequent organ development.
the morphogenetic aspects of gastrulation vary considerably across different groups of organisms.
in general, cells in an outer embryo layer move inward to form the mesoderm and the endoderm, while simultaneously large-scale cell movements and changes in cell shape transform overall embryo structure  <cit> .
a defining feature of gastrulation in amniotes  is that mesoderm cells arise from the epithelial epiblast through an emt in the primitive streak  <cit> .
this contrasts with mesoderm development in lower vertebrates such as frogs and fish in which presumptive mesodermal cells involute and migrate as a generally contiguous sheet.
in chicken, the primitive streak arises following dramatic polonaise cell movements within the epiblast, leading to cell intercalation in the preingression epiblast region  <cit> .
primitive streak formation and the emergence of endoderm and mesoderm is closely integrated with changes in cell fate.
both processes are regulated by several growth factor signalling pathways, including the canonical and non-canonical wnt, pdgf, bmp, nodal, and fgf pathways  <cit> .
in situ hybridization  analyses have shown that members of multiple signalling pathways are expressed in the primitive streak regions of gastrula stage chicken embryos  <cit> .
some of these pathways, as well as other mechanisms, regulate cell migration in the primitive streak  <cit> .
fgf signalling is an important mediator of mesoderm induction and gastrulation movements.
fgfs can induce mesoderm in frog animal caps and avian epiblast  <cit> .
mouse embryos lacking fgfr <dig> initially form a streak, but cells fail to undergo emt due to the absence of snai <dig> expression and failure to downregulate e-cadherin  <cit> .
the downregulation of e-cadherin via transcriptional repression by snail proteins is considered a prerequisite for emt in many contexts  <cit> , including during mouse gastrulation  <cit> .
in chicken embryos, fgfr <dig> signalling is necessary for the primitive streak to form  <cit> .
following emergence of mesoderm cells from the primitive streak, fgfs appear to act as chemotactic factors that influence mesoderm migration.
mesoderm cells will migrate towards a source of fgf <dig> but away from fgf <dig>  <cit> .
in mouse embryos lacking fgf <dig>  emerging mesoderm cells gastrulate but fail to migrate away from the primitive streak  <cit> .
together, these findings indicate that fgf signalling plays a primary role in regulating primitive streak formation, mesoderm induction, and mesoderm migration.
in this study, we investigate how fgf signalling and its downstream effectors regulate cell movement and gene expression in and around the primitive streak of chicken embryos after the onset of gastrulation.
in contrast to results of genetic ablation studies in mice  <cit> , pharmacological inhibition of fgfr activity blocks migration of cells through the primitive streak of chicken embryos by mechanisms that appear to be independent of e-cadherin localization or expression levels.
e-cadherin protein levels are high throughout the epiblast, in cells undergoing emt, and in the newly formed mesoderm, and are unaffected by over expression of snail.
fgfr inhibition leads to downregulation of a large number of regulatory and effector genes through both the ras/mapk and pi3k/akt pathways.
RESULTS
regulatory gene expression in gastrula stage chicken embryos
to obtain an overview of regulatory gene expression patterns in the primitive streak, stage  <dig> embryos were assayed by ish for expression of a candidate group of transcription factors, growth factors, and receptors.
analysis of whole embryos and transverse embryo sections identified several patterns that can be described by combinatorial expression in one or more of the following morphological domains: lateral epiblast, preingression epiblast, primitive streak, medial mesoderm, and lateral mesoderm .
for example, fgfr <dig> is expressed in the lateral epiblast, the preingression epiblast, and the primitive streak, but at greatly reduced levels in the newly formed mesoderm .
fgfr <dig> and fgfr <dig> transcripts are detected in the lateral epiblast but at much lower levels in the preingression epiblast and primitive streak .
genes such as epha <dig>  fgf <dig>  fgf <dig>  pdgfra, and dll <dig> are expressed in the preingression epiblast and primitive streak, and then downregulated in the mesoderm .
snai <dig> shows a similar expression pattern except that transcripts persist to more lateral regions of the mesoderm .
t, wnt5b, wnt8a, and notch <dig> are expressed in the preingression and more lateral epiblast, the primitive streak, and the mesoderm extending to the lateral regions .
finally, genes such as efnb <dig> are expressed in the primitive streak and broadly in the mesoderm .
fgf signalling is required for cell migration through the primitive streak
several observations suggest that fgf signalling is active in the preingression epiblast and primitive streak.
first, fgf <dig>  fgf <dig>  fgf <dig>  fgf <dig>  and fgf <dig> are expressed in these domains .
second, fgfr <dig> transcripts are detected in the lateral and preingression epiblast and in the primitive streak, but at low or undetectable levels in the emerging mesoderm .
fgfr <dig> and fgfr <dig> are expressed at high levels in the lateral epiblast but at much lower or undetectable levels in the preingression epiblast, primitive streak, and mesoderm .
fgfr <dig> transcripts are detected only in extraembryonic regions  <cit> .
third, activated erk , an indicator of fgf signalling, is detected in the preingression epiblast and the primitive streak, with much lower or undetectable levels in emerging mesoderm  <cit> .
while rna localization may not reflect protein expression, these results nevertheless suggest that the fgfr <dig> receptor is present and active in the primitive streak.
to determine if fgfr activity is required for cell migration through the primitive streak, stage 3d- <dig> embryos were pretreated for two hours with the fgfr inhibitor su <dig>  <cit>  or with dmso as a control, and then electroporated with a gfp expression plasmid  <cit> .
extensive control experiments have shown that this electroporation protocol specifically targets cells in the epiblast  <cit> , and so assaying for gfp-positive cells in the mesoderm following a period of development reflects the ability of cells to move from the epiblast through the primitive streak.
the concentration of su <dig> used  was determined by preliminary titration studies to assess the minimum concentration that would abolish detectable expression of t  by ish and phospho-erk by western blot.
analysis of gfp-positive cells in control embryos five hours following electroporation showed typical migration patterns of cells through the primitive streak .
gfp-positive cells were distributed in the lateral and preingression epiblast, primitive streak, and mesoderm layers.
in contrast, gfp-positive cells in su5402-treated embryos were present in the epiblast and primitive streak regions but were rarely observed in the mesoderm layer .
cell counts indicated that significantly more positive cells were retained in the epiblast  and primitive streak  in su <dig> versus dmso treated embryos, while contribution to the mesoderm was virtually abolished by su <dig> .
this data indicates that fgfr activity is required for cells to transition from the epiblast through the primitive streak to populate the mesoderm.
fgf receptor activity is necessary for regulatory gene expression in the primitive streak
components of numerous pathways require fgfr activity for expression.
expression of the receptors pdgfra, epha <dig>  and notch <dig> , and the ligands dll <dig>  wnt5b, wnt8a, and fgf <dig> , were significantly reduced in embryos exposed to su <dig>  surprisingly, fgf <dig> mrna levels were unchanged or slightly elevated by su <dig> treatment .
the t-box transcription factor t was downregulated in the primitive streak but not in hensen's node or the notochord, , while expression of tbx <dig> was globally downregulated by su <dig> treatment .
whereas snai <dig> was expressed in the preingression epiblast, primitive streak, and mesoderm in control embryos , su <dig> treatment inhibited snai <dig> expression only in the preingression epiblast .
the snail transcription factors are widely regarded as key regulators of emt through their ability to downregulate e-cadherin in epithelial cells  <cit> .
in mice, embryos lacking fgfr <dig> fail to express snai <dig> in the primitive streak, leading to the persistence of e-cadherin expression and failure of cells to exit the epiblast and migrate through the primitive streak  <cit> .
since su <dig> abrogates snai <dig> expression only in the preingression epiblast , we investigated the effects of su <dig> treatment on e-cadherin mrna and protein levels and localization.
in control embryos, e-cadherin protein was localized primarily to the periphery of all cells in the epiblast, primitive streak, and medial mesoderm .
e-cadherin labelling in ventral streak cells remained high, while mesodermal cells near the streak showed slightly reduced e-cadherin staining intensity that remained primarily localized to the cell periphery .
in posterior regions of control embryos, e-cadherin labelling was observed throughout the mesoderm layer, while in more anterior regions, e-cadherin levels were reduced in the lateral mesoderm .
surprisingly, in cells of su <dig> treated embryos, neither the levels nor the localization of e-cadherin protein appeared different from controls .
although e-cadherin labelling patterns were indistinguishable between control and su <dig> treated embryos , striking differences in epiblast cell morphology were apparent between the groups.
cells in the preingression epiblast of control embryos exhibited the typical, highly polarized epithelial morphology .
however, in the posterior half of su <dig> treated embryos, cells in the preingression epiblast lacked the characteristic columnar epithelial morphology seen in normal preingression epiblast cells .
in control embryos, n-cadherin protein was detected in all cells of the mesoderm and endoderm layers .
in posterior regions of control embryos, n-cadherin was absent from dorsal primitive streak cells, while in more anterior regions staining was evident in some cells of the dorsal primitive streak .
the relative proportions of n- and e-cadherin labelling varied between individual cells of the streak and the mesoderm layer .
in contrast to e-cadherin, n-cadherin labelling intensity was significantly reduced in the posterior mesoderm of su <dig> treated embryos compared with control embryos .
in agreement with this, ish analysis showed a significant reduction of n-cadherin mrna in the posterior primitive streak region of su <dig> treated embryos .
in more anterior regions, however, n-cadherin staining appeared roughly equivalent in control and su <dig> treated embryos .
n-cadherin labelling intensity was also roughly equivalent in the endoderm of control versus treated embryos .
fgfr inhibition leads to snai <dig> downregulation in the preingression epiblast, but not in the middle to lower portions of the streak or in mesoderm cells .
since snai <dig> is known to repress e-cadherin gene transcription, e-cadherin mrna levels were also assessed by ish and pcr analyses.
by ish, e-cadherin mrna levels in control and su <dig> treated embryos appeared no different in the preingression epiblast or in other regions of the embryo .
by rt-pcr, e-cadherin mrna levels were not statistically different in primitive streak or in preingression epiblast regions of control versus treated embryos , though mrna levels in the isolated preingression epiblast of su <dig> treated embryos showed a trend towards being reduced .
e-cadherin mrna levels in the mesoderm were low but detectable in both control and treated embryos .
altogether, the immunofluorescence, ish and rt-pcr analyses fail to show an increase in e-cadherin levels following inhibition of fgfr activity, despite a reduction of snai <dig> mrna in the preingression epiblast.
to further explore the relationship between snai <dig> and e-cadherin expression, the effect of snail over expression on e-cadherin protein levels and localization was investigated.
three flag-tagged snail expression vectors were utilized: wild-type chicken snai <dig> , wild-type human snai <dig> , and a degradation resistant form of human snai <dig>  that shows an enhanced ability to downregulate e-cadherin and induce emt  <cit> .
the epiblast and primitive streak of stage 3d embryos was electroporated with one of the snail expression vectors , then incubated for  <dig> hours.
following fixation, embryos were assayed by dual immunofluorescence with antibodies to flag or gfp, and to e-cadherin.
regardless of the snail construct used, over expression did not alter e-cadherin protein levels or localization .
snail-positive cells were scattered throughout the epiblast, primitive streak and mesoderm in distributions that were not different from control embryos electroporated with a gfp expression plasmid .
regardless of the snail construct electroporated, over expression did not apparently cause epiblast cells to undergo precocious emt, since many snail-positive cells were observed in the epiblast and in the primitive streak.
importantly, none of the snail constructs appeared to downregulate e-cadherin protein, as snail-expressing cells retained e-cadherin protein at their periphery in patterns indistinguishable from non-expressing cells or cells of embryos expressing gfp .
the ability of snail expression constructs to downregulate e-cadherin was confirmed by transfection into mdck cells .
these results indicate that, within the time-course of the experiment, snail over expression is insufficient to downregulate e-cadherin protein levels.
fgf signalling during gastrulation is propagated via ras/mapk and pi3k/atk pathways
fgf signalling can activate a number of downstream signalling cascades, including ras to mitogen-activated protein kinase  and phosphatidylinositol 3-kinase   <cit> .
to determine if su <dig> affects either of these pathways, the phosphorylation states of erk1/ <dig> and akt were assayed in the preingression epiblast and primitive streak regions of control versus su <dig> treated embryos.
compared with dmso treated control embryos, su <dig> treatment markedly reduced the phosphorylation levels of erk and akt .
to determine the contribution of the ras/mapk and pi3k/akt activity to gene expression in the primitive streak region, embryos were treated with the mek inhibitor u <dig> or the akt inhibitor ly <dig>  and assayed for expression of candidate genes.
consistent with results obtained using su <dig>  inhibition of mek activity resulted in downregulation of the signalling pathway receptors pdgfra, epha <dig>  and notch <dig> , ligands dll <dig>  wnt5b, and wnt8a , and the transcription factor t .
in contrast, fgf <dig> and tbx <dig> transcript levels were unaffected by u <dig> .
as observed following su <dig> treatment, u <dig> abolished snai <dig> expression only in the preingression epiblast while expression in the primitive streak and mesoderm was unaffected .
western blot analysis showed that u <dig> reduced the levels of phosphorylated erk, while levels of phosphorylated akt were unchanged .
the expression of most fgfr and mek-dependent genes was unaffected by akt inhibition .
however, epha <dig>  which was abolished by both su <dig> and u <dig> treatments , and snai <dig>  which was inhibited only in the preingression epiblast region , were also downregulated by ly <dig> .
fgf <dig> and tbx <dig> transcript levels were unaffected by u <dig> treatment , but were essentially abolished by treatment with ly <dig> .
western blot analysis demonstrated that ly <dig> treatment greatly reduced phosphorylated akt levels while levels of phosphorylated erk were unchanged .
together, these results suggest that fgf signalling acts through both erk and akt to control regulatory gene expression in the preingression epiblast and primitive streak.
most fgfr dependent genes assayed required only erk signalling for high-level expression, while expression of a few genes was dependent on signalling only through akt.
epha <dig> expression and snai <dig> expression in the preingression epiblast were dependent on both signalling pathways.
microarray analysis of gene expression
considering the specific effects of fgfr, mek, and akt inhibition on snai <dig> expression in the preingression epiblast, microarray studies were performed to obtain a more comprehensive view of gene expression changes in the different epiblast expression domains illustrated in figure 1a.
first, gene expression levels were compared between lateral and preingression epiblast, excluding the primitive streak.
approximately  <dig> genes were upregulated in the preingression epiblast versus lateral epiblast .
genes upregulated in the preingression epiblast comprised members of several signalling pathways, including notch <dig>  dll <dig>  wnt3a, wnt5b, wnt8a, ednrb, ednrb <dig>  pdgfra, fgf <dig>  fgf <dig>  fgf <dig>  fgf <dig>  and epha <dig>  also upregulated were numerous modulators of fgf signalling, including spry <dig>  spry <dig>  spred <dig>  spred  <dig>  and dusp <dig>  upregulated transcription factor genes included the t-box genes t, tbx <dig>  tbx <dig> and eomes, the ets factors etv <dig>  etv <dig> and elk <dig>  several homeobox-containing genes such as dlx <dig> and mkx, as well as snai <dig>  zic <dig>  atf <dig>  atf <dig>  xbp <dig>  and pou3f <dig> .
u: gene expression increased.
d: gene expressed reduced.
nc: no change.
nd: not determined
superscripts: result confirmed by:  <dig>  microarray;  <dig>  pcr;  <dig>  in situ hybridization
a second series of microarray studies were performed to extend the ish studies above in identifying changes in gene expression in preingression epiblast  of control embryos versus embryos treated with su <dig>  u <dig>  or ly <dig> .
pairwise comparisons of mrna levels in preingression epiblast from control versus treated embryos showed that expression levels of more than  <dig> genes were downregulated in the preingression epiblast following inhibition of fgfr kinase activity by su <dig> treatment .
using gene ontology terms to identify regulatory molecules, fgf signalling was found to regulate numerous ligands, receptors and pathway modulators of several signalling pathways .
of the five fgf ligands expressed in the primitive streak and preingression epiblast, four  were downregulated by su <dig> treatment while fgf <dig> was expressed at control levels.
expression of core components of the fgf signalling pathway was generally unaffected, while numerous positive and negative fgf signalling modulators were downregulated.
members of both the canonical and non-canonical wnt pathways were also downregulated, as were numerous negative regulators of wnt signalling .
expression of rhoa and jnk was also dependent on fgfr activity.
consistent with ish results presented above, the notch <dig> receptor and dll <dig> ligand were highly downregulated, as were the notch pathway transcriptional co repressor ctbp and the co activator snw <dig>  additional signalling pathway genes downregulated by fgfr inhibition included ednrb, ednrb <dig>  pdgfra, and epha <dig>  further confirmation of these results was obtained by realtime rt-pcr analysis .
comparison of expression changes in the preingression epiblast obtained with su <dig>  u <dig>  and ly <dig> revealed several patterns of gene regulation .
most but not all of the genes downregulated by su <dig> were also downregulated by u <dig>  this included all identified fgf pathway members except for fgf <dig> which was unaffected by u <dig>  and fgf <dig> and fgfr <dig> which were unaffected by either inhibitor.
expression of the three ets factors etv <dig>  etv <dig> and elk <dig> was also dependent on mapk signalling.
expression of some transcription factors within the same family showed a differential response to the two inhibitors.
for example, within the t-box transcription factor family, t and eomes were highly downregulated by u <dig> treatment, while tbx <dig> and tbx <dig> transcript levels were not affected.
similarly, the homeobox containing genes dlx <dig> and mkx were unaffected by u <dig> despite being downregulated by su <dig>  in fact, expression of tbx <dig>  tbx <dig>  dlx <dig>  and mkx required pi3k signalling, while being independent of the mapk pathway.
a few genes required signalling through both pathways for expression .
discussion
the role of fgf signalling in regulating gastrulation has been investigated in several classes of organisms.
in frogs, fgf ligands can induce mesoderm in animal cap assays, and fgfr function and downstream pathway activity is required for mesoderm formation  <cit> .
fgfs can induce mesoderm in chicken epiblast  <cit> , and inhibition of fgf signalling blocks appearance of the primitive streak  <cit> .
in mice, fgfr <dig> null embryos form a primitive streak, however primitive streak cells fail to express snai <dig>  to downregulate e-cadherin, or to undergo emt  <cit> .
in the present study, we find that blocking fgfr activity during gastrulation in chicken embryos also inhibits cell migration through the primitive streak.
however, e-cadherin expression is not increased in su <dig> treated embryos, even in the preingression epiblast where snai <dig> expression is reduced.
although loss of e-cadherin is a primary requirement for emt in numerous contexts, immunofluoresence analyses presented here and by others  <cit>  show that emt during avian gastrulation is not temporally linked with downregulation or altered intracellular localization of e-cadherin protein.
emt during gastrulation is closely associated with upregulation of n-cadherin in emerging mesoderm and endoderm cells, while e-cadherin protein levels decline only gradually as cells move to lateral regions of the embryo.
e-cadherin levels also gradually decline in the presomitic mesoderm.
over expression of three different snail proteins also failed to alter e-cadherin protein levels or localization in epiblast, primitive streak, or mesoderm cells.
these findings do not rule out more subtle changes in e-cadherin function in the primitive streak unrelated to protein expression levels or localization detectable by confocal microscopy.
the temporally controlled pharmacological approach used in this study may not be directly comparable to the fgfr gene ablation studies reported in mice  <cit> .
nevertheless, it appears that there are significant differences between chickens and mice in the regulatory pathways downstream of fgf signalling controlling the movement of cells through the primitive streak.
evidence presented here suggests that fgf-dependent pathways controlling emt are independent of changes in e-cadherin expression, and furthermore that loss of e-cadherin is not temporally associated with emt.
how, then, is fgf signalling regulating the movement of cells through the chicken embryo primitive streak?
the broad requirement of fgf signalling for expression of components of numerous regulatory pathways during avian gastrulation suggests that fgf signalling may co-ordinately control multiple pathways related to the emt process.
rhoa regulation of microtubule dynamics is required to regulate basement membrane breakdown and emt during avian gastrulation  <cit> , and non-canonical wnt signalling is necessary for cells to transition from epiblast through the primitive streak to the mesoderm  <cit> .
while direct regulation of these pathways by fgf signalling has not been addressed, expression of pathway components  is regulated by fgfr activity .
expression of the epha receptor, epha <dig>  in preingression epiblast and primitive streak is also dependent on fgf signalling.
preliminary studies indicate that its function is also necessary for cells to undergo gastrulation .
n-cadherin expression is also dependent on fgfr activity.
recent findings have shown that n-cadherin expression is required for cells to properly migrate away from the primitive streak  <cit> .
the intracellular fgf signalling antagonists sproutys and spreds contribute to the coordinate regulation of mesoderm induction and cell movement by differentially regulating signalling downstream of the fgf receptor-ligand interaction.
sproutys antagonize plcγ signalling to regulate convergent extension, while spreds regulate the ras/mapk pathway to modulate gene expression  <cit> .
in the chicken preingression epiblast, fgfr/ras/mapk signalling regulates expression of sprouty <dig>  sprouty <dig>  sprouty <dig>  and spred <dig>  suggesting that both pathways are activated.
although roles for notch, endothelin and canonical wnt pathways in regulating emt during avian gastrulation have not yet been delineated, major components of each pathway are regulated by fgf signalling and each has been shown to regulate aspects of emt in other contexts  <cit> .
it is intriguing that all fgf ligands examined except fgf <dig> are downregulated following inhibition of fgf signalling.
mechanisms regulating its expression are not known.
several laboratories have shown that fgf <dig> is required for mesoderm cells to migrate away from the primitive streak, but not for emt.
mouse embryos lacking fgf <dig>  show normal emt within the primitive streak, however mesodermal cells fail to migrate away from the midline  <cit> .
it is possible that both ligands regulate the migration of mesoderm cells, because, in chicken, the lateral migration of mesoderm cells is directed towards a source of fgf <dig> but away from fgf <dig>  <cit> .
how cells achieve this directional migration is unclear.
emerging mesoderm cells downregulate fgf receptor expression , although they become re-expressed as cells move to more lateral regions.
fgfr activity in the preingression epiblast controls both the ras/mapk and pi3k/akt pathways.
some genes are regulated only through one pathway, while a few genes require both pathways for expression.
for example, t expression is mediated through ras/mapk but is independent of pi3k/akt signalling, while the related t-box factors tbx <dig> and tbx <dig> require pi3k/akt signalling but are independent of the ras/mapk pathway.
the t-box factor eomes requires both pathways for expression.
the regulation of snai <dig> expression by fgfr signalling is particularly interesting.
following inhibition of fgfr kinase activity , snai <dig> expression is downregulated in the preingression epiblast but not in the primitive streak or mesoderm.
this finding supports the concept of modular regulation of gene expression in the primitive streak region , and also highlights species-specific differences in the regulation of snail genes.
collectively, the fgfr inhibitor su <dig>  the mek inhibitor u <dig>  and pi3k inhibitor ly <dig> have been used in a large number of published studies to investigate fgf signalling pathways.
however, in some contexts, each can inhibit other pathways, and multiple signalling pathways can signal through mek and/or pi3k.
su <dig>  for example, can also inhibit the activity of vegfr <dig>  in this study, it is highly unlikely that vegf signalling rather than fgf signalling is regulating the pathways shown to be affected by su <dig>  because vegfr <dig> is not expressed in the avian primitive streak and the vegfr inhibitors su <dig> and su <dig> fail to reduce expression of tbx <dig> or t in the primitive streak .
since mek and akt can act downstream of pathways other than fgf signalling, we have limited our comparisons of genes regulated by u <dig> and ly <dig> to those that are also regulated by su <dig>
although in this study we have focused on genes that are positively regulated by fgf signalling, the preingression epiblast-specific downregulation of snai <dig> indicates that another likely function of fgf signalling is to repress gene expression in cells moving from the lateral to preingression epiblast.
at least  <dig> genes are downregulated in the preingression epiblast versus lateral epiblast, and at least  <dig> of these genes are upregulated following su <dig> treatment.
snai <dig>  and perhaps other transcriptional repressors, might also function to repress the transcription of genes that would preclude transition from epiblast to mesoderm and endoderm.
these transcription factors may also repress genes that are upregulated by gastrulation signals but whose precocious expression prior to entering the streak would be detrimental.
while additional studies will be required to identify the underlying biological significance, fgf signalling in the preingression epiblast both activates and represses gene expression.
CONCLUSIONS
we have shown that fgf signalling is required for the movement of cells from the epiblast through the primitive streak to the mesoderm of gastrula stage chicken embryos.
fgf dependent mechanisms regulating migration are independent of apparent alterations in e-cadherin protein expression or localization.
further, the levels and intracellular localization of e-cadherin do not appear to change as cells undergo emt during gastrulation.
fgf signalling positively and negatively regulates the expression of a large number of genes in the preingression epiblast, primitive streak and newly formed mesoderm layer .
these include members of several major signalling pathways, among them the fgf, canonical and non-canonical wnt, notch, pdgf, eph-ephrin, and endothelin pathways.
a large number of transcriptional regulatory factors are also regulated by fgfr activity, and well as the cell adhesion molecule, n-cadherin.
of pathways known to regulate cell migration through the primitive streak, fgf signalling regulates the expression of components of several, including rhoa and non-canonical wnt5b.

 vertebrate gastrulation is a highly coordinated process that leads to formation of the three primary germ layers and sets up the body plan for subsequent organ development. the morphogenetic aspects of gastrulation vary considerably across different groups of organisms. in general, cells in an outer embryo layer move inward to form the mesoderm and the endoderm, while simultaneously large-scale cell movements and changes in cell shape transform overall embryo structure. a defining feature of gastrulation in amniotes is that mesoderm cells arise from the epithelial epiblast through an emt in the primitive streak. this contrasts with mesoderm development in lower vertebrates such as frogs and fish in which presumptive mesodermal cells involute and migrate as a generally contiguous sheet. in chicken, the primitive streak arises following dramatic polonaise cell movements within the epiblast, leading to cell intercalation in the preingression epiblast region. primitive streak formation and the emergence of endoderm and mesoderm is closely integrated with changes in cell fate. both processes are regulated by several growth factor signalling pathways, including the canonical and non-canonical wnt, pdgf, bmp, nodal, and fgf pathways. in situ hybridization analyses have shown that members of multiple signalling pathways are expressed in the primitive streak regions of gastrula stage chicken embryos. some of these pathways, as well as other mechanisms, regulate cell migration in the primitive streak. fgf signalling is an important mediator of mesoderm induction and gastrulation movements. fgfs can induce mesoderm in frog animal caps and avian epiblast. mouse embryos lacking fgfr initially form a streak, but cells fail to undergo emt due to the absence of snai expression and failure to downregulate e-cadherin. the downregulation of e-cadherin via transcriptional repression by snail proteins is considered a prerequisite for emt in many contexts, including during mouse gastrulation. in chicken embryos, fgfr signalling is necessary for the primitive streak to form. following emergence of mesoderm cells from the primitive streak, fgfs appear to act as chemotactic factors that influence mesoderm migration. mesoderm cells will migrate towards a source of fgf but away from fgf. in mouse embryos lacking fgf emerging mesoderm cells gastrulate but fail to migrate away from the primitive streak. together, these findings indicate that fgf signalling plays a primary role in regulating primitive streak formation, mesoderm induction, and mesoderm migration. in this study, we investigate how fgf signalling and its downstream effectors regulate cell movement and gene expression in and around the primitive streak of chicken embryos after the onset of gastrulation. in contrast to results of genetic ablation studies in mice, pharmacological inhibition of fgfr activity blocks migration of cells through the primitive streak of chicken embryos by mechanisms that appear to be independent of e-cadherin localization or expression levels. e-cadherin protein levels are high throughout the epiblast, in cells undergoing emt, and in the newly formed mesoderm, and are unaffected by over expression of snail. fgfr inhibition leads to downregulation of a large number of regulatory and effector genes through both the ras/mapk and pi3k/akt pathways.  regulatory gene expression in gastrula stage chicken embryos to obtain an overview of regulatory gene expression patterns in the primitive streak, stage embryos were assayed by ish for expression of a candidate group of transcription factors, growth factors, and receptors. analysis of whole embryos and transverse embryo sections identified several patterns that can be described by combinatorial expression in one or more of the following morphological domains: lateral epiblast, preingression epiblast, primitive streak, medial mesoderm, and lateral mesoderm. for example, fgfr is expressed in the lateral epiblast, the preingression epiblast, and the primitive streak, but at greatly reduced levels in the newly formed mesoderm. fgfr and fgfr transcripts are detected in the lateral epiblast but at much lower levels in the preingression epiblast and primitive streak. genes such as epha fgf fgf pdgfra, and dll are expressed in the preingression epiblast and primitive streak, and then downregulated in the mesoderm. snai shows a similar expression pattern except that transcripts persist to more lateral regions of the mesoderm. t, wnt5b, wnt8a, and notch are expressed in the preingression and more lateral epiblast, the primitive streak, and the mesoderm extending to the lateral regions. finally, genes such as efnb are expressed in the primitive streak and broadly in the mesoderm. fgf signalling is required for cell migration through the primitive streak several observations suggest that fgf signalling is active in the preingression epiblast and primitive streak. first, fgf fgf fgf fgf and fgf are expressed in these domains. second, fgfr transcripts are detected in the lateral and preingression epiblast and in the primitive streak, but at low or undetectable levels in the emerging mesoderm. fgfr and fgfr are expressed at high levels in the lateral epiblast but at much lower or undetectable levels in the preingression epiblast, primitive streak, and mesoderm. fgfr transcripts are detected only in extraembryonic regions. third, activated erk, an indicator of fgf signalling, is detected in the preingression epiblast and the primitive streak, with much lower or undetectable levels in emerging mesoderm. while rna localization may not reflect protein expression, these results nevertheless suggest that the fgfr receptor is present and active in the primitive streak. to determine if fgfr activity is required for cell migration through the primitive streak, stage 3d- embryos were pretreated for two hours with the fgfr inhibitor su or with dmso as a control, and then electroporated with a gfp expression plasmid. extensive control experiments have shown that this electroporation protocol specifically targets cells in the epiblast, and so assaying for gfp-positive cells in the mesoderm following a period of development reflects the ability of cells to move from the epiblast through the primitive streak. the concentration of su used was determined by preliminary titration studies to assess the minimum concentration that would abolish detectable expression of t by ish and phospho-erk by western blot. analysis of gfp-positive cells in control embryos five hours following electroporation showed typical migration patterns of cells through the primitive streak. gfp-positive cells were distributed in the lateral and preingression epiblast, primitive streak, and mesoderm layers. in contrast, gfp-positive cells in su5402-treated embryos were present in the epiblast and primitive streak regions but were rarely observed in the mesoderm layer. cell counts indicated that significantly more positive cells were retained in the epiblast and primitive streak in su versus dmso treated embryos, while contribution to the mesoderm was virtually abolished by su. this data indicates that fgfr activity is required for cells to transition from the epiblast through the primitive streak to populate the mesoderm. fgf receptor activity is necessary for regulatory gene expression in the primitive streak components of numerous pathways require fgfr activity for expression. expression of the receptors pdgfra, epha and notch, and the ligands dll wnt5b, wnt8a, and fgf, were significantly reduced in embryos exposed to su surprisingly, fgf mrna levels were unchanged or slightly elevated by su treatment. the t-box transcription factor t was downregulated in the primitive streak but not in hensen's node or the notochord,, while expression of tbx was globally downregulated by su treatment. whereas snai was expressed in the preingression epiblast, primitive streak, and mesoderm in control embryos, su treatment inhibited snai expression only in the preingression epiblast. the snail transcription factors are widely regarded as key regulators of emt through their ability to downregulate e-cadherin in epithelial cells. in mice, embryos lacking fgfr fail to express snai in the primitive streak, leading to the persistence of e-cadherin expression and failure of cells to exit the epiblast and migrate through the primitive streak. since su abrogates snai expression only in the preingression epiblast, we investigated the effects of su treatment on e-cadherin mrna and protein levels and localization. in control embryos, e-cadherin protein was localized primarily to the periphery of all cells in the epiblast, primitive streak, and medial mesoderm. e-cadherin labelling in ventral streak cells remained high, while mesodermal cells near the streak showed slightly reduced e-cadherin staining intensity that remained primarily localized to the cell periphery. in posterior regions of control embryos, e-cadherin labelling was observed throughout the mesoderm layer, while in more anterior regions, e-cadherin levels were reduced in the lateral mesoderm. surprisingly, in cells of su treated embryos, neither the levels nor the localization of e-cadherin protein appeared different from controls. although e-cadherin labelling patterns were indistinguishable between control and su treated embryos, striking differences in epiblast cell morphology were apparent between the groups. cells in the preingression epiblast of control embryos exhibited the typical, highly polarized epithelial morphology. however, in the posterior half of su treated embryos, cells in the preingression epiblast lacked the characteristic columnar epithelial morphology seen in normal preingression epiblast cells. in control embryos, n-cadherin protein was detected in all cells of the mesoderm and endoderm layers. in posterior regions of control embryos, n-cadherin was absent from dorsal primitive streak cells, while in more anterior regions staining was evident in some cells of the dorsal primitive streak. the relative proportions of n- and e-cadherin labelling varied between individual cells of the streak and the mesoderm layer. in contrast to e-cadherin, n-cadherin labelling intensity was significantly reduced in the posterior mesoderm of su treated embryos compared with control embryos. in agreement with this, ish analysis showed a significant reduction of n-cadherin mrna in the posterior primitive streak region of su treated embryos. in more anterior regions, however, n-cadherin staining appeared roughly equivalent in control and su treated embryos. n-cadherin labelling intensity was also roughly equivalent in the endoderm of control versus treated embryos. fgfr inhibition leads to snai downregulation in the preingression epiblast, but not in the middle to lower portions of the streak or in mesoderm cells. since snai is known to repress e-cadherin gene transcription, e-cadherin mrna levels were also assessed by ish and pcr analyses. by ish, e-cadherin mrna levels in control and su treated embryos appeared no different in the preingression epiblast or in other regions of the embryo. by rt-pcr, e-cadherin mrna levels were not statistically different in primitive streak or in preingression epiblast regions of control versus treated embryos, though mrna levels in the isolated preingression epiblast of su treated embryos showed a trend towards being reduced. e-cadherin mrna levels in the mesoderm were low but detectable in both control and treated embryos. altogether, the immunofluorescence, ish and rt-pcr analyses fail to show an increase in e-cadherin levels following inhibition of fgfr activity, despite a reduction of snai mrna in the preingression epiblast. to further explore the relationship between snai and e-cadherin expression, the effect of snail over expression on e-cadherin protein levels and localization was investigated. three flag-tagged snail expression vectors were utilized: wild-type chicken snai, wild-type human snai, and a degradation resistant form of human snai that shows an enhanced ability to downregulate e-cadherin and induce emt. the epiblast and primitive streak of stage 3d embryos was electroporated with one of the snail expression vectors, then incubated for hours. following fixation, embryos were assayed by dual immunofluorescence with antibodies to flag or gfp, and to e-cadherin. regardless of the snail construct used, over expression did not alter e-cadherin protein levels or localization. snail-positive cells were scattered throughout the epiblast, primitive streak and mesoderm in distributions that were not different from control embryos electroporated with a gfp expression plasmid. regardless of the snail construct electroporated, over expression did not apparently cause epiblast cells to undergo precocious emt, since many snail-positive cells were observed in the epiblast and in the primitive streak. importantly, none of the snail constructs appeared to downregulate e-cadherin protein, as snail-expressing cells retained e-cadherin protein at their periphery in patterns indistinguishable from non-expressing cells or cells of embryos expressing gfp. the ability of snail expression constructs to downregulate e-cadherin was confirmed by transfection into mdck cells. these results indicate that, within the time-course of the experiment, snail over expression is insufficient to downregulate e-cadherin protein levels. fgf signalling during gastrulation is propagated via ras/mapk and pi3k/atk pathways fgf signalling can activate a number of downstream signalling cascades, including ras to mitogen-activated protein kinase and phosphatidylinositol 3-kinase. to determine if su affects either of these pathways, the phosphorylation states of erk1/ and akt were assayed in the preingression epiblast and primitive streak regions of control versus su treated embryos. compared with dmso treated control embryos, su treatment markedly reduced the phosphorylation levels of erk and akt. to determine the contribution of the ras/mapk and pi3k/akt activity to gene expression in the primitive streak region, embryos were treated with the mek inhibitor u or the akt inhibitor ly and assayed for expression of candidate genes. consistent with results obtained using su inhibition of mek activity resulted in downregulation of the signalling pathway receptors pdgfra, epha and notch, ligands dll wnt5b, and wnt8a, and the transcription factor t. in contrast, fgf and tbx transcript levels were unaffected by u. as observed following su treatment, u abolished snai expression only in the preingression epiblast while expression in the primitive streak and mesoderm was unaffected. western blot analysis showed that u reduced the levels of phosphorylated erk, while levels of phosphorylated akt were unchanged. the expression of most fgfr and mek-dependent genes was unaffected by akt inhibition. however, epha which was abolished by both su and u treatments, and snai which was inhibited only in the preingression epiblast region, were also downregulated by ly. fgf and tbx transcript levels were unaffected by u treatment, but were essentially abolished by treatment with ly. western blot analysis demonstrated that ly treatment greatly reduced phosphorylated akt levels while levels of phosphorylated erk were unchanged. together, these results suggest that fgf signalling acts through both erk and akt to control regulatory gene expression in the preingression epiblast and primitive streak. most fgfr dependent genes assayed required only erk signalling for high-level expression, while expression of a few genes was dependent on signalling only through akt. epha expression and snai expression in the preingression epiblast were dependent on both signalling pathways. microarray analysis of gene expression considering the specific effects of fgfr, mek, and akt inhibition on snai expression in the preingression epiblast, microarray studies were performed to obtain a more comprehensive view of gene expression changes in the different epiblast expression domains illustrated in figure 1a. first, gene expression levels were compared between lateral and preingression epiblast, excluding the primitive streak. approximately genes were upregulated in the preingression epiblast versus lateral epiblast. genes upregulated in the preingression epiblast comprised members of several signalling pathways, including notch dll wnt3a, wnt5b, wnt8a, ednrb, ednrb pdgfra, fgf fgf fgf fgf and epha also upregulated were numerous modulators of fgf signalling, including spry spry spred spred and dusp upregulated transcription factor genes included the t-box genes t, tbx tbx and eomes, the ets factors etv etv and elk several homeobox-containing genes such as dlx and mkx, as well as snai zic atf atf xbp and pou3f. u: gene expression increased. d: gene expressed reduced. nc: no change. nd: not determined superscripts: result confirmed by: microarray; pcr; in situ hybridization a second series of microarray studies were performed to extend the ish studies above in identifying changes in gene expression in preingression epiblast of control embryos versus embryos treated with su u or ly. pairwise comparisons of mrna levels in preingression epiblast from control versus treated embryos showed that expression levels of more than genes were downregulated in the preingression epiblast following inhibition of fgfr kinase activity by su treatment. using gene ontology terms to identify regulatory molecules, fgf signalling was found to regulate numerous ligands, receptors and pathway modulators of several signalling pathways. of the five fgf ligands expressed in the primitive streak and preingression epiblast, four were downregulated by su treatment while fgf was expressed at control levels. expression of core components of the fgf signalling pathway was generally unaffected, while numerous positive and negative fgf signalling modulators were downregulated. members of both the canonical and non-canonical wnt pathways were also downregulated, as were numerous negative regulators of wnt signalling. expression of rhoa and jnk was also dependent on fgfr activity. consistent with ish results presented above, the notch receptor and dll ligand were highly downregulated, as were the notch pathway transcriptional co repressor ctbp and the co activator snw additional signalling pathway genes downregulated by fgfr inhibition included ednrb, ednrb pdgfra, and epha further confirmation of these results was obtained by realtime rt-pcr analysis. comparison of expression changes in the preingression epiblast obtained with su u and ly revealed several patterns of gene regulation. most but not all of the genes downregulated by su were also downregulated by u this included all identified fgf pathway members except for fgf which was unaffected by u and fgf and fgfr which were unaffected by either inhibitor. expression of the three ets factors etv etv and elk was also dependent on mapk signalling. expression of some transcription factors within the same family showed a differential response to the two inhibitors. for example, within the t-box transcription factor family, t and eomes were highly downregulated by u treatment, while tbx and tbx transcript levels were not affected. similarly, the homeobox containing genes dlx and mkx were unaffected by u despite being downregulated by su in fact, expression of tbx tbx dlx and mkx required pi3k signalling, while being independent of the mapk pathway. a few genes required signalling through both pathways for expression. discussion the role of fgf signalling in regulating gastrulation has been investigated in several classes of organisms. in frogs, fgf ligands can induce mesoderm in animal cap assays, and fgfr function and downstream pathway activity is required for mesoderm formation. fgfs can induce mesoderm in chicken epiblast, and inhibition of fgf signalling blocks appearance of the primitive streak. in mice, fgfr null embryos form a primitive streak, however primitive streak cells fail to express snai to downregulate e-cadherin, or to undergo emt. in the present study, we find that blocking fgfr activity during gastrulation in chicken embryos also inhibits cell migration through the primitive streak. however, e-cadherin expression is not increased in su treated embryos, even in the preingression epiblast where snai expression is reduced. although loss of e-cadherin is a primary requirement for emt in numerous contexts, immunofluoresence analyses presented here and by others show that emt during avian gastrulation is not temporally linked with downregulation or altered intracellular localization of e-cadherin protein. emt during gastrulation is closely associated with upregulation of n-cadherin in emerging mesoderm and endoderm cells, while e-cadherin protein levels decline only gradually as cells move to lateral regions of the embryo. e-cadherin levels also gradually decline in the presomitic mesoderm. over expression of three different snail proteins also failed to alter e-cadherin protein levels or localization in epiblast, primitive streak, or mesoderm cells. these findings do not rule out more subtle changes in e-cadherin function in the primitive streak unrelated to protein expression levels or localization detectable by confocal microscopy. the temporally controlled pharmacological approach used in this study may not be directly comparable to the fgfr gene ablation studies reported in mice. nevertheless, it appears that there are significant differences between chickens and mice in the regulatory pathways downstream of fgf signalling controlling the movement of cells through the primitive streak. evidence presented here suggests that fgf-dependent pathways controlling emt are independent of changes in e-cadherin expression, and furthermore that loss of e-cadherin is not temporally associated with emt. how, then, is fgf signalling regulating the movement of cells through the chicken embryo primitive streak? the broad requirement of fgf signalling for expression of components of numerous regulatory pathways during avian gastrulation suggests that fgf signalling may co-ordinately control multiple pathways related to the emt process. rhoa regulation of microtubule dynamics is required to regulate basement membrane breakdown and emt during avian gastrulation, and non-canonical wnt signalling is necessary for cells to transition from epiblast through the primitive streak to the mesoderm. while direct regulation of these pathways by fgf signalling has not been addressed, expression of pathway components is regulated by fgfr activity. expression of the epha receptor, epha in preingression epiblast and primitive streak is also dependent on fgf signalling. preliminary studies indicate that its function is also necessary for cells to undergo gastrulation. n-cadherin expression is also dependent on fgfr activity. recent findings have shown that n-cadherin expression is required for cells to properly migrate away from the primitive streak. the intracellular fgf signalling antagonists sproutys and spreds contribute to the coordinate regulation of mesoderm induction and cell movement by differentially regulating signalling downstream of the fgf receptor-ligand interaction. sproutys antagonize plcγ signalling to regulate convergent extension, while spreds regulate the ras/mapk pathway to modulate gene expression. in the chicken preingression epiblast, fgfr/ras/mapk signalling regulates expression of sprouty sprouty sprouty and spred suggesting that both pathways are activated. although roles for notch, endothelin and canonical wnt pathways in regulating emt during avian gastrulation have not yet been delineated, major components of each pathway are regulated by fgf signalling and each has been shown to regulate aspects of emt in other contexts. it is intriguing that all fgf ligands examined except fgf are downregulated following inhibition of fgf signalling. mechanisms regulating its expression are not known. several laboratories have shown that fgf is required for mesoderm cells to migrate away from the primitive streak, but not for emt. mouse embryos lacking fgf show normal emt within the primitive streak, however mesodermal cells fail to migrate away from the midline. it is possible that both ligands regulate the migration of mesoderm cells, because, in chicken, the lateral migration of mesoderm cells is directed towards a source of fgf but away from fgf. how cells achieve this directional migration is unclear. emerging mesoderm cells downregulate fgf receptor expression, although they become re-expressed as cells move to more lateral regions. fgfr activity in the preingression epiblast controls both the ras/mapk and pi3k/akt pathways. some genes are regulated only through one pathway, while a few genes require both pathways for expression. for example, t expression is mediated through ras/mapk but is independent of pi3k/akt signalling, while the related t-box factors tbx and tbx require pi3k/akt signalling but are independent of the ras/mapk pathway. the t-box factor eomes requires both pathways for expression. the regulation of snai expression by fgfr signalling is particularly interesting. following inhibition of fgfr kinase activity, snai expression is downregulated in the preingression epiblast but not in the primitive streak or mesoderm. this finding supports the concept of modular regulation of gene expression in the primitive streak region, and also highlights species-specific differences in the regulation of snail genes. collectively, the fgfr inhibitor su the mek inhibitor u and pi3k inhibitor ly have been used in a large number of published studies to investigate fgf signalling pathways. however, in some contexts, each can inhibit other pathways, and multiple signalling pathways can signal through mek and/or pi3k. su for example, can also inhibit the activity of vegfr in this study, it is highly unlikely that vegf signalling rather than fgf signalling is regulating the pathways shown to be affected by su because vegfr is not expressed in the avian primitive streak and the vegfr inhibitors su and su fail to reduce expression of tbx or t in the primitive streak. since mek and akt can act downstream of pathways other than fgf signalling, we have limited our comparisons of genes regulated by u and ly to those that are also regulated by su although in this study we have focused on genes that are positively regulated by fgf signalling, the preingression epiblast-specific downregulation of snai indicates that another likely function of fgf signalling is to repress gene expression in cells moving from the lateral to preingression epiblast. at least genes are downregulated in the preingression epiblast versus lateral epiblast, and at least of these genes are upregulated following su treatment. snai and perhaps other transcriptional repressors, might also function to repress the transcription of genes that would preclude transition from epiblast to mesoderm and endoderm. these transcription factors may also repress genes that are upregulated by gastrulation signals but whose precocious expression prior to entering the streak would be detrimental. while additional studies will be required to identify the underlying biological significance, fgf signalling in the preingression epiblast both activates and represses gene expression.  we have shown that fgf signalling is required for the movement of cells from the epiblast through the primitive streak to the mesoderm of gastrula stage chicken embryos. fgf dependent mechanisms regulating migration are independent of apparent alterations in e-cadherin protein expression or localization. further, the levels and intracellular localization of e-cadherin do not appear to change as cells undergo emt during gastrulation. fgf signalling positively and negatively regulates the expression of a large number of genes in the preingression epiblast, primitive streak and newly formed mesoderm layer. these include members of several major signalling pathways, among them the fgf, canonical and non-canonical wnt, notch, pdgf, eph-ephrin, and endothelin pathways. a large number of transcriptional regulatory factors are also regulated by fgfr activity, and well as the cell adhesion molecule, n-cadherin. of pathways known to regulate cell migration through the primitive streak, fgf signalling regulates the expression of components of several, including rhoa and non-canonical wnt5b. 
we find that pharmacological inhibition of fgfr activity blocks migration of cells through the primitive streak of chicken embryos without apparent alterations in the level or intracellular localization of e-cadherin. fgfr inhibition leads to downregulation of a large number of regulatory genes in the preingression epiblast adjacent to the primitive streak, the primitive streak and the newly formed mesoderm. this includes members of the fgf, notch, eph, pdgf, and canonical and non-canonical wnt pathways, negative modulators of these pathways, and a large number of transcriptional regulatory genes. snai expression in the primitive streak and mesoderm is not altered by fgfr inhibition, but is downregulated only in the preingression epiblast region with no significant effect on e-cadherin. pharmacological inhibition of mek or akt indicate that these downstream effectors control discrete and overlapping groups of genes during gastrulation. the positive and negative effects on large groups of genes by pharmacological inhibition of fgf signalling, including major signalling pathways and transcription factor families, indicates that the fgf pathway is a focal point of regulation during gastrulation in chicken. in mice lacking fgfr epiblast cells in the primitive streak fail to downregulate e-cadherin and undergo emt, and cell migration is inhibited. fgfr activity regulates components of several pathways known to be required for cell migration through the streak or in the mesoderm, including rhoa, the non-canonical wnt pathway, pdgf signalling and the cell adhesion protein n-cadherin. during gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition in the primitive streak to form the mesoderm and endoderm. this study investigated how fgf signalling regulates cell movement and gene expression in the primitive streak of chicken embryos. e-cadherin protein is localized to the periphery of epiblast, primitive streak and some mesodermal cells. furthermore, over expression of snail has no discernable effect on e-cadherin protein levels or localization in epiblast, primitive streak or mesodermal cells. in chicken embryos, fgf signalling regulates cell movement through the primitive streak by mechanisms that appear to be independent of changes in e-cadherin expression or protein localization.
 fgf signalling regulates numerous aspects of early embryo development. during gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition in the primitive streak to form the mesoderm and endoderm. in mice lacking fgfr epiblast cells in the primitive streak fail to downregulate e-cadherin and undergo emt, and cell migration is inhibited. this study investigated how fgf signalling regulates cell movement and gene expression in the primitive streak of chicken embryos.  we find that pharmacological inhibition of fgfr activity blocks migration of cells through the primitive streak of chicken embryos without apparent alterations in the level or intracellular localization of e-cadherin. e-cadherin protein is localized to the periphery of epiblast, primitive streak and some mesodermal cells. fgfr inhibition leads to downregulation of a large number of regulatory genes in the preingression epiblast adjacent to the primitive streak, the primitive streak and the newly formed mesoderm. this includes members of the fgf, notch, eph, pdgf, and canonical and non-canonical wnt pathways, negative modulators of these pathways, and a large number of transcriptional regulatory genes. snai expression in the primitive streak and mesoderm is not altered by fgfr inhibition, but is downregulated only in the preingression epiblast region with no significant effect on e-cadherin. furthermore, over expression of snail has no discernable effect on e-cadherin protein levels or localization in epiblast, primitive streak or mesodermal cells. fgfr activity modulates distinct downstream pathways including ras/mapk and pi3k/akt. pharmacological inhibition of mek or akt indicate that these downstream effectors control discrete and overlapping groups of genes during gastrulation. fgfr activity regulates components of several pathways known to be required for cell migration through the streak or in the mesoderm, including rhoa, the non-canonical wnt pathway, pdgf signalling and the cell adhesion protein n-cadherin.  in chicken embryos, fgf signalling regulates cell movement through the primitive streak by mechanisms that appear to be independent of changes in e-cadherin expression or protein localization. the positive and negative effects on large groups of genes by pharmacological inhibition of fgf signalling, including major signalling pathways and transcription factor families, indicates that the fgf pathway is a focal point of regulation during gastrulation in chicken. 