BACKGROUND
the definitive endoderm  gives rise to the gastrointestinal and digestive system.
in the mouse embryo, the de progenitors reside at the posterior region of the epiblast that derived from the inner cell mass  <cit> .
during gastrulation, as the cells ingress through the primitive streak, the epiblast segregates into the three germ layers that form the somatic cell lineages of the ectoderm, mesoderm, and definitive endoderm .
de arises from the forkhead box a <dig> -expressing anterior primitive streak   and is then regionalized into the fore-, mid-, and hindgut  <cit> .
the de is identified by the expression of sry -box  <dig>   and chemokine  receptor  <dig>  .
sox <dig> mutant mouse embryos have a reduced de, apoptosis of the foregut, and abnormal morphogenesis of the mid- and hindgut  <cit> .
sox <dig> is also required for the assembly of the basement membrane, as the sox <dig> mutant embryo fails to segregate the de from the mesoderm  <cit> .
activin is a frequently used inducer for de differentiation from pluripotent stem cells, embryonic stem cells , and induced pluripotent stem cells  .
when sox <dig> is overexpressed, human escs spontaneously differentiate into the de, independent of activin  <cit> .
in zebrafish embryos, cxcr4a regulates directional migration  <cit>  and de proliferation during gastrulation  <cit> .
in chick embryos, cxcr <dig> is expressed in the de and angioblasts.
cxcr <dig> and its ligand cxcl <dig> form a reciprocal signaling loop that triggers angioblast migration to the pancreatic endoderm and induces pancreatic development.
inhibition of cxcr <dig> suppresses angioblast migration into the pancreatic endoderm region.
as a result, the size of the pancreas decreases  <cit> .
although cxcr <dig> is also expressed in the mesodermal cells, it is often used in combination with e-cadherin for purifying esc-derived de cells using flow cytometry  <cit> .
daf <dig> is an inhibitor of complementary activation  <cit> .
daf <dig> is expressed by immune cells and de-derived tissue, such as intestine and airway  <cit> .
using microarray analysis and in situ hybridization, we previously identified daf <dig> as a de cell surface marker based on its expression in esc-derived and embryonic de.
daf <dig> is also expressed in pancreatic progenitor cells  <cit> .
however, the role of daf <dig> in the de is not well understood.
in this study, we found that the de population that expresses daf <dig>  has slow cell cycling and low cell-matrix adhesive characteristics.
furthermore, daf1-negative de cells  turn out to be foxa2 + sox17low cells and daf1-positive de  cells to be foxa2 + sox17high cells.
our results therefore suggest that e-cadherin + cxcr4 + de is composed of two populations: sox17low early de and sox17high late de.
sox17high late de cells were positive for daf <dig>  and were slow proliferative and low cell-matrix adhesive cells.
RESULTS
daf1 + de are slowly proliferating cells
previously, we reported daf <dig> as a surface marker, expressed in a subpopulation of de  <cit> .
de are defined as e-cadherin+/cxcr4+ cells  <cit> .
when cultured in activin-containing medium  <cit> , escs sequentially give rise to aps cells on day <dig> , then to de cells  on day  <dig> .
a plot of our previous microarray analysis results of the aps and de cells  <cit>  shows the time dependent expression of foxa <dig>  sox <dig> and daf <dig> .
foxa <dig> was highly expressed in the aps and de.
sox <dig> was lowly expressed in the aps and highly expressed in the de.
daf <dig> expression was absent in the aps and present in the de .
we then analyzed daf1+ cells for the expression of nanog, a pluripotent marker, using a mouse nanog-ips cell line bearing a green fluorescent protein  reporter driven under nanog promoter.
daf1-positive cells turned out to be nanog/gfp negative .
taken together, daf <dig> marks a subpopulation of de and is first expressed in the de, but not earlier in the aps.fig.
<dig> daf1+ de cells are less proliferative than daf1- de cells.
a mouse escs  were differentiated first to the anterior primitive streak then into the definitive endoderm .
our previously reported microarray analysis of foxa <dig>  sox <dig> and daf <dig> expression during de differentiation are plotted in a time dependent manner.
daf <dig> expression increased in the de but not in the aps.
esc; mouse embryonic stem cells, aps; anterior primitive streak, de; definitive endoderm.
b schematic drawing of the experiment to analyze cell cycle phases of the sorted de.
es cells were differentiated into de, then dissociated and sorted for daf1+/- cells.
the sorted cells were immediately analyzed for cell cycle.
c real time-pcr analysis showed that daf <dig> expression was enriched in daf1+ de.
y axis shows relative gene expression, with  <dig> equivalent to daf <dig> expression level in daf1 + de cells.
d, e proliferative properties of daf1 + de and daf1-de cells were assayed.
d cell proliferation marker, ph <dig> , and pcna  was down-regulated in daf1 + de.
analyzed by western blot analysis.
e cell cycle analysis revealed that daf1 + de reside longer in the g0/g <dig> phase with a shorter s and m/g <dig> phases compared to that of the daf1-de cells.
raw data are shown in additional file  <dig>  student’s two tailed t-test
to identify the differences between daf1 + de and daf1-de cells, we purified daf1+/-de cells and compared their properties.
real time pcr analysis of the sorted daf1+/-de cells confirmed daf <dig> expression is enriched in daf1 + de .
expressions of an m phase marker, phosphorylated histone h <dig>  or proliferating cell nuclear antigen  that marks proliferating cells at every phase of the cell except g <dig> were enriched in daf1-de than in daf1 + de cells, revealed by western blot analysis .
we analyzed the cell cycle phases and found that daf1 + de cells showed a longer go/g <dig> phase and shorter s, m/g <dig> phase compared to daf1-de cells .
this suggested that cell proliferation was decreased in daf1 + de.
daf1 + de are low adhesive cells
next, we performed flow cytometry using antibody against daf1and plated at a same cell number of the sorted de cells  onto matrigel-pre-coated dishes .
after 24 h culture, the daf1+ de gave rise to fewer cell number compared to that of daf1-de .
the results suggested a higher level of cell death and/or lower plating efficiency of daf1 + de cells.
we then examined cell-matrix adhesion and the extent of apoptosis, in which cells were allowed to adhere to the plate for  <dig> mins  <cit>   <cit> .
our results revealed that daf1 + de cells showed a lower matrix adhesion ratio than daf1-de , and that daf1 + de cells were more apoptotic and showed a higher cleaved caspase3/7+ ratio  than daf1-de.
cells bind to extracellular matrix  through the ecm receptor, integrin  <cit> .
we found that expression of several subtypes of the integrin genes, such as itgα <dig>  itgα <dig>  and itgα <dig>  was down-regulated in daf1 + de cells .
our results suggest that daf1 + de cells have lower adhesion capacity than daf1-de cells, which led to a lower plating efficiency and triggered cell death in the attached cells, compared to daf1-de cells.fig.
<dig> daf1+ de cells have lower cell-matrix adhesion than daf1- de cells.
a a schematic drawing showing experimental design.
the sorted de cells were plated onto matrigel-pre-coated dishes and cultured for 24 h  or 90 min .
b transmission images of the cells derived from the daf1+/-de cells after cultured for 24 h. c after 24 h culture, the daf1 + de cells gave rise to fewer cells.
d cell-matrix adhesion assay, after 90 min culture.
quantification of the attached cells.
the adhesion ability of daf1 + de cells onto matrigel is weaker than that of the daf1-de cells.
e quantification of cleaved caspase3/7+ apoptotic cells.
the daf1 + de cells gave rise to a larger proportion of apoptotic cells.
f the integrin expression profiles of the de cells detected by rt-pcr analysis.
student’s two tailed t-test.
scale bars; 100 μm
daf <dig> is a marker of late stage de
to characterize the adherent cells onto matrigel- pre-coated dish, we assayed for the expression of the de markers, foxa <dig> and sox <dig>  immediately after the adhesion assay .
interestingly, whereas both daf1+/-de populations showed similar capacity for giving rise to foxa2-expressing cells, daf1 + de cells showed a higher capacity for giving rise to sox17-expressing cells .
foxa <dig> is expressed in both the aps and de, whereas sox <dig> is expressed at a higher level in the de.
we then confirmed sox <dig> protein expression of the sorted daf1+/-de cells directly  by immunocytochemical analysis .
sox <dig> expression was found in a higher proportion and seemingly higher level in the sorted daf1 + de cells compared to daf1-de cells .
western blot analysis confirmed a higher level of sox <dig> expression in the daf1 + de cells .
since foxa <dig> expression is detected prior to that of sox <dig>  we then asked if daf1-de cells represent early de and daf1 + de cells represent late de, by examining if daf1-de cells differentiate into daf1 + de.
daf1+/-de cells sorted by flow cytometry  were re-cultured on mouse embryonic fibroblast  feeders with activin containing endoderm differentiation medium.
we examined the expression of daf <dig>  to exclude mef feeders, we analyzed e-cadherin + cells.
almost all of the daf1-de cells acquired daf <dig> expression over the course of 24 h and gave rise to daf1 + de , whereas daf1 + de cells did not turn into daf1-de .
taken together, these results suggest that daf1-de represent early and daf1 + de represent late de cells during de differentiation and that daf1-de become daf1 + de cells .fig.
<dig> daf1-de is the progenitor of daf1+ de.
a a schematic drawing showing experimental design.
b quantifications of foxa2+ and sox17+ cells by immunocytochemistry after 90 min cell-matrix adhesion assay.
c, d the proportion of cells expressing sox <dig> was higher in the daf1 + de as detected by immunocytochemical analysis , and the level of sox <dig> expression was higher in the daf1 + de as detected by western blot , of the sorted de cells .
e flow cytometry analysis of the descendent cells of daf1-de and daf1 + de.
left panels: daf <dig>  cxcr <dig> expression profiles of the sorted daf1+/- de cells.
right panels: both daf1-de and daf1 + de acquired daf <dig> expression after 24 h. f the scheme of fig.
<dig>  sox17low daf1-de differentiated into sox17high daf1 + de.
student’s two tailed t-test.
scale bar; 100 μm
daf1+/-de cells can give rise to pancreatic and intestinal fates
we identified that daf1-de cells acquired daf <dig> expression and turned into daf1 + de.
if daf1-de represents early de and daf1 + de represents late de, both de cells could give rise to regional endoderm derivatives of the gut.
we next examined the abilities of the daf1+/-de cells to differentiate into pancreatic or intestinal cells.
to visualize pancreatic differentiation, we used pdx1/gfp mouse escs, in which gfp expression is driven by the pdx <dig> promoter  <cit> .
we isolated daf1+/-de cells by flow cytometry , seeded the cells on mef feeders, and cultured in the presence of activin and fgf <dig>   <cit> .
we found that both daf1- de and daf1+ cells gave rise to pdx1/gfp+ cells, after plated for 3 days .
the daf1 + de-derived cells yielded less pdx1/gfp cells compared to that of the daf1-de cells .
this discrepancy was probably due to a lower proliferation of the daf1 + de cells compared to daf1-de cells.
since the pancreas derives from the anterior de, we then asked if the daf1+/-de cells could also differentiate into the posterior de derivative of the intestine.
de derived from r <dig> escs were sorted and seeded onto mef feeders and cultured at the presence of bio and dapt  <cit> .
after five days, both daf1-de and daf1 + de cells differentiated into cdx2+ intestinal cells.
similar to pancreatic differentiation, daf1 + de cells are less proliferative and gave rise to small cdx2+ colonies than daf1-de cells .
our results therefore suggest that both daf1+/- des are capable of differentiating into the endodermal derivatives of the pancreas and intestine.fig.
<dig> both daf1+/- could differentiate into pancreatic and intestinal fates.
a a schematic drawing showing experimental design.
de was sorted on day  <dig>  the sorted cells were plated onto mef feeders and cultured with pancreatic and intestinal differentiation medium.
daf1- or daf1 + de cells differentiated into pdx1/gfp+ cells after 4 days culture  and cdx2+ cells after 5 days culture .
b arrows depict small colonies of daf1 + de-derived pdx1/gfp+ cells.
numbers depict differentiation ratios: daf1-de-derived pdx1/gfp+ cells;  <dig>  %, daf1 + de-derived pdx1/gfp+ cells; 12 % .
c numbers depict differentiation ratios: daf1-de-derived cdx2+ cells;  <dig>  %, daf1 + de-derived cdx2+ cells;  <dig>  % .
scale bar; 100 μm
fig.
<dig> proposed role of daf <dig> in de differentiation.
icm/esc differentiated into the e-cadherin + cxcr4 + foxa2 + sox17lowdaf1- early de.
these cells then differentiated into the e-cadherin + cxcr4 + foxa2 + sox17highdaf1+ late de.
the late de differentiated into regionalized anterior  and posterior  endodermal lineages.
transition from the daf1-de to daf1 + de is accompanied by restricted cell proliferation and cell-matrix adhesion.
icm; inner cell mass
discussion
previously, we identified that daf <dig> is expressed in the de.
here, we identified daf <dig> as a late de marker.
daf <dig> is an inhibitor of complement activation  <cit> .
daf <dig> is expressed in the kidney, spleen, testis, intestine, and bronchi of the adult mouse  <cit> .
daf <dig> deficiency is reported in autoimmune hemolytic anemia patients  <cit> .
in daf <dig> knockout mice, ifn-γ expression increases, resulting in enhanced t cell response autoimmunity  <cit> .
however, gastrointestinal-tract develops normally in daf <dig> knockout mice.
here, we examined the detailed expression patterns during de differentiation using escs.
de cells are defined as e-cadherin+/cxcr4+ cells  <cit> .
however, both e-cadherin and cxcr <dig> are also expressed in the primitive streak  <cit> .
therefore, the use of e-cadherin+/cxcr4+ as a marker to define the de cells is confined to a limited time window.
moreover, e-cadherin+/cxcr4+ de cells are a heterogeneous population.
here we used daf <dig> to characterize a subpopulation of e-cadherin+/cxcr4+ de cells.
we revealed that both daf1-de cells represent early de and daf1 + de represent late de.
daf1-de and daf1 + de cells can give rise to the pancreatic and intestinal lineages.
daf1 + de formed small colonies, due to their less proliferative and low adhesive characteristics than that of daf1-de cells .
a slight decrease in s, m/g <dig> phase and increase in g0/g <dig> phase in daf1 + de cells might reflect their property as more differentiated cells.
daf1 + de cells seem to differentiate as efficiently into pdx1-expressing cells, but not as efficiently into cdx2-expressing cells, compared to daf1-de cells.
this might due to a partial loss in differentiation potency of daf1+ de cells into the intestinal fate.
we previously reported that regional-specific endodermal fates are determined sequentially in the order of stomach, intestine and pancreas, in the chick embryos  <cit> .
it is possible that daf1 + de gradually lose potency to differentiate into intestinal lineages, but retains differentiation potency into pancreatic lineages, compared to that of daf1-de.
cell-matrix adhesion is also necessary for cell differentiation  <cit> .
integrin expression promotes de differentiation from human pluripotent stem cells  <cit> .
integrin is a receptor of extracellular matrix expressed in the cells, which enables binding of the cells to the extracellular matrix.
itgα <dig> and itgαv are de-specific integrins.
knockdown of either itgα <dig> or itgαv inhibits de differentiation  <cit> .
both daf1- and daf1 + de cells express itgα <dig> and itgαv.
we found that the expressions of itgα <dig>  itgα <dig>  and itgα <dig> decreased in daf1 + de cells.
itgα <dig> is an attachment molecule of the de  <cit> .
itgα <dig> is expressed in the de and expression decreases in the foxa <dig> null mouse embryo  <cit> .
itgα <dig> null mice have abnormal lung morphogenesis  <cit> .
these daf1-de specific integrins could regulate de differentiation and modulate their behavior.
the integrins are known to show distinct ligand binding specificities among the superfamily members.
itgα1β <dig>  itgα3β <dig> bind specifically to laminin and itgα8β <dig> binds specifically to fibronectin  <cit> .
the lowered expression of itgα <dig>  itgα <dig> might explain the decreased adhesion of daf1 + de cells to matrigel, which composed mainly of laminin.
the decrease in adhesion to matrix might reflect the developmental transition from early to late de.
we found that daf1-de could turn into daf1 + de cells.
sox <dig> expression was higher in daf1 + de than in daf1-de cells.
furthermore, expression of a primitive streak marker, brachyury  <cit> , was higher in daf1-de cells .
therefore, e-cadherin+/cxcr4+ de could be a mixed population of both primitive streak and de cells.
CONCLUSIONS
our data indicate that de can be divided into two stages: early and late des.
early de consists of e-cadherin + cxcr4 + daf1-foxa2 + sox17low cells that show higher proliferative activity and higher cell-matrix adhesive capacity.
later on, these de cells differentiate into e-cadherin + cxcr4 + daf1+ foxa2 + sox17high late de cells that show a decreased proliferation and low cell-matrix adhesion capacity.
our findings would contribute to the understandings of the differentiation of the primitive streak and de during gastrulation.

 the definitive endoderm gives rise to the gastrointestinal and digestive system. in the mouse embryo, the de progenitors reside at the posterior region of the epiblast that derived from the inner cell mass. during gastrulation, as the cells ingress through the primitive streak, the epiblast segregates into the three germ layers that form the somatic cell lineages of the ectoderm, mesoderm, and definitive endoderm. de arises from the forkhead box a -expressing anterior primitive streak and is then regionalized into the fore-, mid-, and hindgut. the de is identified by the expression of sry -box and chemokine receptor. sox mutant mouse embryos have a reduced de, apoptosis of the foregut, and abnormal morphogenesis of the mid- and hindgut. sox is also required for the assembly of the basement membrane, as the sox mutant embryo fails to segregate the de from the mesoderm. activin is a frequently used inducer for de differentiation from pluripotent stem cells, embryonic stem cells, and induced pluripotent stem cells. when sox is overexpressed, human escs spontaneously differentiate into the de, independent of activin. in zebrafish embryos, cxcr4a regulates directional migration and de proliferation during gastrulation. in chick embryos, cxcr is expressed in the de and angioblasts. cxcr and its ligand cxcl form a reciprocal signaling loop that triggers angioblast migration to the pancreatic endoderm and induces pancreatic development. inhibition of cxcr suppresses angioblast migration into the pancreatic endoderm region. as a result, the size of the pancreas decreases. although cxcr is also expressed in the mesodermal cells, it is often used in combination with e-cadherin for purifying esc-derived de cells using flow cytometry. daf is an inhibitor of complementary activation. daf is expressed by immune cells and de-derived tissue, such as intestine and airway. using microarray analysis and in situ hybridization, we previously identified daf as a de cell surface marker based on its expression in esc-derived and embryonic de. daf is also expressed in pancreatic progenitor cells. however, the role of daf in the de is not well understood. in this study, we found that the de population that expresses daf has slow cell cycling and low cell-matrix adhesive characteristics. furthermore, daf1-negative de cells turn out to be foxa2 + sox17low cells and daf1-positive de cells to be foxa2 + sox17high cells. our results therefore suggest that e-cadherin + cxcr4 + de is composed of two populations: sox17low early de and sox17high late de. sox17high late de cells were positive for daf and were slow proliferative and low cell-matrix adhesive cells.  daf1 + de are slowly proliferating cells previously, we reported daf as a surface marker, expressed in a subpopulation of de. de are defined as e-cadherin+/cxcr4+ cells. when cultured in activin-containing medium, escs sequentially give rise to aps cells on day, then to de cells on day. a plot of our previous microarray analysis results of the aps and de cells shows the time dependent expression of foxa sox and daf. foxa was highly expressed in the aps and de. sox was lowly expressed in the aps and highly expressed in the de. daf expression was absent in the aps and present in the de. we then analyzed daf1+ cells for the expression of nanog, a pluripotent marker, using a mouse nanog-ips cell line bearing a green fluorescent protein reporter driven under nanog promoter. daf1-positive cells turned out to be nanog/gfp negative. taken together, daf marks a subpopulation of de and is first expressed in the de, but not earlier in the aps.fig. daf1+ de cells are less proliferative than daf1- de cells. a mouse escs were differentiated first to the anterior primitive streak then into the definitive endoderm. our previously reported microarray analysis of foxa sox and daf expression during de differentiation are plotted in a time dependent manner. daf expression increased in the de but not in the aps. esc; mouse embryonic stem cells, aps; anterior primitive streak, de; definitive endoderm. b schematic drawing of the experiment to analyze cell cycle phases of the sorted de. es cells were differentiated into de, then dissociated and sorted for daf1+/- cells. the sorted cells were immediately analyzed for cell cycle. c real time-pcr analysis showed that daf expression was enriched in daf1+ de. y axis shows relative gene expression, with equivalent to daf expression level in daf1 + de cells. d, e proliferative properties of daf1 + de and daf1-de cells were assayed. d cell proliferation marker, ph, and pcna was down-regulated in daf1 + de. analyzed by western blot analysis. e cell cycle analysis revealed that daf1 + de reside longer in the g0/g phase with a shorter s and m/g phases compared to that of the daf1-de cells. raw data are shown in additional file student’s two tailed t-test to identify the differences between daf1 + de and daf1-de cells, we purified daf1+/-de cells and compared their properties. real time pcr analysis of the sorted daf1+/-de cells confirmed daf expression is enriched in daf1 + de. expressions of an m phase marker, phosphorylated histone h or proliferating cell nuclear antigen that marks proliferating cells at every phase of the cell except g were enriched in daf1-de than in daf1 + de cells, revealed by western blot analysis. we analyzed the cell cycle phases and found that daf1 + de cells showed a longer go/g phase and shorter s, m/g phase compared to daf1-de cells. this suggested that cell proliferation was decreased in daf1 + de. daf1 + de are low adhesive cells next, we performed flow cytometry using antibody against daf1and plated at a same cell number of the sorted de cells onto matrigel-pre-coated dishes. after 24 h culture, the daf1+ de gave rise to fewer cell number compared to that of daf1-de. the results suggested a higher level of cell death and/or lower plating efficiency of daf1 + de cells. we then examined cell-matrix adhesion and the extent of apoptosis, in which cells were allowed to adhere to the plate for mins. our results revealed that daf1 + de cells showed a lower matrix adhesion ratio than daf1-de, and that daf1 + de cells were more apoptotic and showed a higher cleaved caspase3/7+ ratio than daf1-de. cells bind to extracellular matrix through the ecm receptor, integrin. we found that expression of several subtypes of the integrin genes, such as itgα itgα and itgα was down-regulated in daf1 + de cells. our results suggest that daf1 + de cells have lower adhesion capacity than daf1-de cells, which led to a lower plating efficiency and triggered cell death in the attached cells, compared to daf1-de cells.fig. daf1+ de cells have lower cell-matrix adhesion than daf1- de cells. a a schematic drawing showing experimental design. the sorted de cells were plated onto matrigel-pre-coated dishes and cultured for 24 h or 90 min. b transmission images of the cells derived from the daf1+/-de cells after cultured for 24 h. c after 24 h culture, the daf1 + de cells gave rise to fewer cells. d cell-matrix adhesion assay, after 90 min culture. quantification of the attached cells. the adhesion ability of daf1 + de cells onto matrigel is weaker than that of the daf1-de cells. e quantification of cleaved caspase3/7+ apoptotic cells. the daf1 + de cells gave rise to a larger proportion of apoptotic cells. f the integrin expression profiles of the de cells detected by rt-pcr analysis. student’s two tailed t-test. scale bars; 100 μm daf is a marker of late stage de to characterize the adherent cells onto matrigel- pre-coated dish, we assayed for the expression of the de markers, foxa and sox immediately after the adhesion assay. interestingly, whereas both daf1+/-de populations showed similar capacity for giving rise to foxa2-expressing cells, daf1 + de cells showed a higher capacity for giving rise to sox17-expressing cells. foxa is expressed in both the aps and de, whereas sox is expressed at a higher level in the de. we then confirmed sox protein expression of the sorted daf1+/-de cells directly by immunocytochemical analysis. sox expression was found in a higher proportion and seemingly higher level in the sorted daf1 + de cells compared to daf1-de cells. western blot analysis confirmed a higher level of sox expression in the daf1 + de cells. since foxa expression is detected prior to that of sox we then asked if daf1-de cells represent early de and daf1 + de cells represent late de, by examining if daf1-de cells differentiate into daf1 + de. daf1+/-de cells sorted by flow cytometry were re-cultured on mouse embryonic fibroblast feeders with activin containing endoderm differentiation medium. we examined the expression of daf to exclude mef feeders, we analyzed e-cadherin + cells. almost all of the daf1-de cells acquired daf expression over the course of 24 h and gave rise to daf1 + de, whereas daf1 + de cells did not turn into daf1-de. taken together, these results suggest that daf1-de represent early and daf1 + de represent late de cells during de differentiation and that daf1-de become daf1 + de cells.fig. daf1-de is the progenitor of daf1+ de. a a schematic drawing showing experimental design. b quantifications of foxa2+ and sox17+ cells by immunocytochemistry after 90 min cell-matrix adhesion assay. c, d the proportion of cells expressing sox was higher in the daf1 + de as detected by immunocytochemical analysis, and the level of sox expression was higher in the daf1 + de as detected by western blot, of the sorted de cells. e flow cytometry analysis of the descendent cells of daf1-de and daf1 + de. left panels: daf cxcr expression profiles of the sorted daf1+/- de cells. right panels: both daf1-de and daf1 + de acquired daf expression after 24 h. f the scheme of fig.  sox17low daf1-de differentiated into sox17high daf1 + de. student’s two tailed t-test. scale bar; 100 μm daf1+/-de cells can give rise to pancreatic and intestinal fates we identified that daf1-de cells acquired daf expression and turned into daf1 + de. if daf1-de represents early de and daf1 + de represents late de, both de cells could give rise to regional endoderm derivatives of the gut. we next examined the abilities of the daf1+/-de cells to differentiate into pancreatic or intestinal cells. to visualize pancreatic differentiation, we used pdx1/gfp mouse escs, in which gfp expression is driven by the pdx promoter. we isolated daf1+/-de cells by flow cytometry, seeded the cells on mef feeders, and cultured in the presence of activin and fgf. we found that both daf1- de and daf1+ cells gave rise to pdx1/gfp+ cells, after plated for 3 days. the daf1 + de-derived cells yielded less pdx1/gfp cells compared to that of the daf1-de cells. this discrepancy was probably due to a lower proliferation of the daf1 + de cells compared to daf1-de cells. since the pancreas derives from the anterior de, we then asked if the daf1+/-de cells could also differentiate into the posterior de derivative of the intestine. de derived from r escs were sorted and seeded onto mef feeders and cultured at the presence of bio and dapt. after five days, both daf1-de and daf1 + de cells differentiated into cdx2+ intestinal cells. similar to pancreatic differentiation, daf1 + de cells are less proliferative and gave rise to small cdx2+ colonies than daf1-de cells. our results therefore suggest that both daf1+/- des are capable of differentiating into the endodermal derivatives of the pancreas and intestine.fig. both daf1+/- could differentiate into pancreatic and intestinal fates. a a schematic drawing showing experimental design. de was sorted on day the sorted cells were plated onto mef feeders and cultured with pancreatic and intestinal differentiation medium. daf1- or daf1 + de cells differentiated into pdx1/gfp+ cells after 4 days culture and cdx2+ cells after 5 days culture. b arrows depict small colonies of daf1 + de-derived pdx1/gfp+ cells. numbers depict differentiation ratios: daf1-de-derived pdx1/gfp+ cells;  %, daf1 + de-derived pdx1/gfp+ cells; 12 %. c numbers depict differentiation ratios: daf1-de-derived cdx2+ cells;  %, daf1 + de-derived cdx2+ cells;  %. scale bar; 100 μm fig. proposed role of daf in de differentiation. icm/esc differentiated into the e-cadherin + cxcr4 + foxa2 + sox17lowdaf1- early de. these cells then differentiated into the e-cadherin + cxcr4 + foxa2 + sox17highdaf1+ late de. the late de differentiated into regionalized anterior and posterior endodermal lineages. transition from the daf1-de to daf1 + de is accompanied by restricted cell proliferation and cell-matrix adhesion. icm; inner cell mass discussion previously, we identified that daf is expressed in the de. here, we identified daf as a late de marker. daf is an inhibitor of complement activation. daf is expressed in the kidney, spleen, testis, intestine, and bronchi of the adult mouse. daf deficiency is reported in autoimmune hemolytic anemia patients. in daf knockout mice, ifn-γ expression increases, resulting in enhanced t cell response autoimmunity. however, gastrointestinal-tract develops normally in daf knockout mice. here, we examined the detailed expression patterns during de differentiation using escs. de cells are defined as e-cadherin+/cxcr4+ cells. however, both e-cadherin and cxcr are also expressed in the primitive streak. therefore, the use of e-cadherin+/cxcr4+ as a marker to define the de cells is confined to a limited time window. moreover, e-cadherin+/cxcr4+ de cells are a heterogeneous population. here we used daf to characterize a subpopulation of e-cadherin+/cxcr4+ de cells. we revealed that both daf1-de cells represent early de and daf1 + de represent late de. daf1-de and daf1 + de cells can give rise to the pancreatic and intestinal lineages. daf1 + de formed small colonies, due to their less proliferative and low adhesive characteristics than that of daf1-de cells. a slight decrease in s, m/g phase and increase in g0/g phase in daf1 + de cells might reflect their property as more differentiated cells. daf1 + de cells seem to differentiate as efficiently into pdx1-expressing cells, but not as efficiently into cdx2-expressing cells, compared to daf1-de cells. this might due to a partial loss in differentiation potency of daf1+ de cells into the intestinal fate. we previously reported that regional-specific endodermal fates are determined sequentially in the order of stomach, intestine and pancreas, in the chick embryos. it is possible that daf1 + de gradually lose potency to differentiate into intestinal lineages, but retains differentiation potency into pancreatic lineages, compared to that of daf1-de. cell-matrix adhesion is also necessary for cell differentiation. integrin expression promotes de differentiation from human pluripotent stem cells. integrin is a receptor of extracellular matrix expressed in the cells, which enables binding of the cells to the extracellular matrix. itgα and itgαv are de-specific integrins. knockdown of either itgα or itgαv inhibits de differentiation. both daf1- and daf1 + de cells express itgα and itgαv. we found that the expressions of itgα itgα and itgα decreased in daf1 + de cells. itgα is an attachment molecule of the de. itgα is expressed in the de and expression decreases in the foxa null mouse embryo. itgα null mice have abnormal lung morphogenesis. these daf1-de specific integrins could regulate de differentiation and modulate their behavior. the integrins are known to show distinct ligand binding specificities among the superfamily members. itgα1β itgα3β bind specifically to laminin and itgα8β binds specifically to fibronectin. the lowered expression of itgα itgα might explain the decreased adhesion of daf1 + de cells to matrigel, which composed mainly of laminin. the decrease in adhesion to matrix might reflect the developmental transition from early to late de. we found that daf1-de could turn into daf1 + de cells. sox expression was higher in daf1 + de than in daf1-de cells. furthermore, expression of a primitive streak marker, brachyury, was higher in daf1-de cells. therefore, e-cadherin+/cxcr4+ de could be a mixed population of both primitive streak and de cells.  our data indicate that de can be divided into two stages: early and late des. early de consists of e-cadherin + cxcr4 + daf1-foxa2 + sox17low cells that show higher proliferative activity and higher cell-matrix adhesive capacity. later on, these de cells differentiate into e-cadherin + cxcr4 + daf1+ foxa2 + sox17high late de cells that show a decreased proliferation and low cell-matrix adhesion capacity. our findings would contribute to the understandings of the differentiation of the primitive streak and de during gastrulation. 
previously, we identified a novel de marker, decay accelerating factor by identifying de specific genes from the expression profile of de derived from mouse embryonic stem cells by microarray analysis, and in situ hybridization of early embryos. definitive endoderm gives rise to the respiratory apparatus and digestive tract. daf1-expressing late definitive endoderm proliferates slowly and show low adhesive capacity. pluripotent stem celldefinitive endodermdaf1in vitro differentiationproliferationadhesionmext, japangrants-in-aid 26253059kume shoen http://dx.doi.org/ /501100001700ministry of education, culture, sports, science, and technologygrants-in-aid 26670384higo programkume shoen http://dx.doi.org/ /501100003478ministry of health, labour and welfarehealth and labor sciences research grants “research on regulatory science of pharmaceuticals and medical devices”shiraki nobuaki issue-copyright-statement© the author 2016
 definitive endoderm gives rise to the respiratory apparatus and digestive tract. sox and cxcr are useful markers of the de. previously, we identified a novel de marker, decay accelerating factor by identifying de specific genes from the expression profile of de derived from mouse embryonic stem cells by microarray analysis, and in situ hybridization of early embryos. daf is expressed in a subpopulation of e-cadherin + cxcr4+ de cells. the characteristics of the daf1-expressing cells during de differentiation has not been examined.  in this report, we utilized the esc differentiation system to examine the characteristics of daf1-expressing de cells. we found that daf expression could discriminate late de from early de. early de cells are daf1-negative and late de cells are daf1-positive. we also found that daf1+ late de cells show low proliferative and low cell matrix adhesive characteristics. furthermore, the purified sox17low early de cells gave rise to daf1+ sox17high late de cells.  daf1-expressing late definitive endoderm proliferates slowly and show low adhesive capacity. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords pluripotent stem celldefinitive endodermdaf1in vitro differentiationproliferationadhesionmext, japangrants-in-aid 26253059kume shoen http://dx.doi.org/ /501100001700ministry of education, culture, sports, science, and technologygrants-in-aid 26670384higo programkume shoen http://dx.doi.org/ /501100003478ministry of health, labour and welfarehealth and labor sciences research grants “research on regulatory science of pharmaceuticals and medical devices”shiraki nobuaki issue-copyright-statement© the author 2016 