in ovo electroporation and in situ hybridization was performed as in previous studies ( 6 ) , and at least three embryos were analyzed for each condition . rna probes were transcribed in vitro from cdna clones provided by bbsrc ( bmp7 : chest290n8 ; bapx1 : chest177b22 ; pitx2 : chest76c15 ) , except for the neurog3 cdna provided by anne grapin - botton and the bmp4 cdna provided by cliff tabin . probes for nkx6.1 and hes1 were used as in earlier studies ( 6,17 ) the xenopus noggin cdna was provided by richard harland , and a cooh - terminal cmyc epitope tag inserted by pcr . the mouse dnalk3 was a cooh - terminal truncated receptor ( encoding aa 1237 ) lacking the kinase domain . a similar construct has previously been used to block bmp signaling in duck and chicken embryos ( 18 ) . briefly , embryos were cleared in dent 's bleach and incubated overnight with antibodies diluted in blocking buffer . confocal images were obtained from embryos cleared in benzyl alcohol / benzyl benzoate ( babb ) ( 19 ) on a zeiss lsm510 axioimager . for antibodies , these data can be found in online appendix supplementary table 1 , available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1010/dc1 . perfusion of embryos with lens culinaris agglutinin was performed with a protocol modified from jilani et al . the embryos were dissected in pbs , and 10 l was injected into the heart with a pulled glass pipette . labeled areas were quantified using imagej software by analyzing every second optical section corresponding to an optical depth of 20 m through the whole pancreas ( between 7 and 12 sections from each embryos ) . lentiviral constructs were generated by gateway recombination cloning , using entry vectors with the same constructs used in the chicken experiments . ( 21 ) , except that the epithelium was not separated from the mesenchyme before lentiviral infection . all lentiviral vectors were used at viral concentrations corresponding to 300 ng of p24 capsid protein per explant . the e12.5-pancreas explants were fixed in formalin and embedded in paraffin 7 days after transduction . the explants were sectioned ( 4 m ) and stained for insulin , amylase , and dna ( dapi ) as described by haumaitre et al . quantification was performed every fifth section spanning the entire explant using imagej software , and total area , insulin area , and amylase area surface were measured . in the e10.5 gut tube explants 10-m cryosections of the entire explants were stained for insulin , amylase , and dna ( dapi ) . quantification was performed every second section by counting the nuclei of insulin and amylase - positive cells . bmp signaling is transduced by smad1 , 5 , and 8 which , upon signaling , become phosphorylated and accumulates in the nucleus where they act as transcription factors ( 23 ) . we used a phosphosmad1,5,8-specific antibody ( 24 ) to characterize endogenous bmp signaling in the embryonic chicken pancreas ( fig . 1 ) at hamburger and hamilton ( hh ) stages 12 , 18 , and 21 ( 25 ) . this corresponds to time points when the pancreas has just been specified in the endoderm , dorsal and ventral buds have formed , and -cells , -cells , and amylase - positive cells can be detected ( 6,17 ) . psmad1,5,8 stainings on e2 , e3 , and e4 ( hh st.12 , 18 , and 21 ) chicken embryos ( a  j ) , e9.5 mouse embryo ( k  l ) , and e11.5-dissected mouse gut ( m  n ) . images are either projections of confocal z - stacks of thick vibratome sections ( a  c ) or an optical section ( d ) . boxed area in b is shown in higher magnification in c and d. a : projected image from a z - stack showing psmad1,5,8 reactivity in the ventral forgut endoderm ( arrows ) and dorsal neural tube ( arrowhead ) . b : projected image at the presumptive pancreas level . note psmad1,5,8 reactivity in the lateral plate mesoderm and the underlying endoderm that appears yellow because of coexpression with foxa2 ( arrow ) . c and d : psmad1,5,8 reactivity can be observed in the endothelial cells of the dorsal aortas and in endoderm lateral to the nkx6.1 expression domain ( arrowhead ) . e and f : a section of an e3 pancreas stained as indicated ( shown with and without nkx6.1 ) . note the psmad1,5,8 reactivity in the mesenchyme and the absence of staining in the pancreas epithelium ( arrows point to the dorsal pancreas ) . g : composite of two images showing the dorsal and ventral pancreas and the liver ; psmad1,5,8 can be detected in the pancreas mesenchyme and in mesenchymal and epithelial compartments of the liver . h  j : another section from the same pancreas showing absence of psmad1,5,8 immunoreactivity in the nkx6.1-positive pancreatic epithelium and -cells ( recognizable by their morphology , arrowhead ) . k and l : optical sections from a z - stack of an e9.5 mouse stained in whole mount ; psmad1,5,8 can be detected in the dorsal aorta and intersomitic vessels and in mesenchymal cells in close contact with the ventral pancreas epithelium . a , aorta ; dp , dorsal pancreas ; duo , duodenum , v , right omphalomesenteric vein ; vp , ventral pancreas . ( a high - quality digital representation of this figure is available in the online issue . ) at hh st . 12 , embryos were whole - mount stained and 200-m transverse vibratome sections were analyzed ( fig . d ) and in the ventral part of the anterior foregut endoderm ( arrows , fig . 1a , ) as previously reported ( 24 ) . at the level of the 3rd to 8th somite pairs , where the prospective pancreatic endoderm is located ( 3,26 ) , psmad1,5,8 was detected in the lateral plate mesoderm and in the endoderm lateral to the nkx6.1 expression domain ( fig . 1 ) . we could also observe psmad1,5,8 in the endothelium of the paired dorsal aortas ( fig . 18 , psmad1,5,8 immunoreactivity was detected in the pancreas mesenchyme and at lower levels in the duodenal epithelium ( fig . one day later , we still observed a mesenchymal psmad1,5,8 signal ( fig . 1g j ) , but no signal was detected in the dorsal or ventral pancreatic epithelium . the liver epithelium had a strong signal with a sharp border of immunoreactivity in the bile duct ( fig . 1 g arrows ) . no psmad1,5,8 reactivity was detected in nkx6.1-positive cells , which at this stage comprise both undifferentiated epithelial cells and -cells ( 17 ) ( fig . the absence of labeling in -cells was confirmed by colabeling with an insulin antibody ( supplementary fig . 1 g and h ) . to evaluate the status of smad1,5,8 phosphorylation in the early mouse pancreas , we performed whole - mount staining at e9.5 and e11.5 for psmad1,5,8 combined with pdx1 ( fig . no psmad1,5,8 was detected in the pdx1-positive endoderm . at e11.5 , we observed psmad1,5,8 reactivity in the pancreas mesenchyme , but no reactivity in the pancreas epithelium ( fig we conclude that endogenous signaling leading to detectable levels of psmad1,5,8 in the early chicken and mouse pancreas is entirely restricted to the mesenchyme . bmp4 , -5 , and -7 mrna have been reported to be expressed in the embryonic mouse pancreas by rt - pcr ( 15 ) , and bmp4 and bmp7 have been localized to the pancreas epithelium ( 16 ) , but little is known about bmp expression during chicken pancreas development . 22 ) to characterize the expression patterns of bmp2 , bmp4 , bmp5 , and bmp7 ( fig . 2 and data not shown ) . we found that bmp4 and bmp7 are expressed in the chicken pancreas mesenchyme and excluded from the epithelium at the stages examined ( fig . we could not detect bmp2 or bmp5 in the pancreas at any stage ( data not shown ) . from these results we concluded that bmp signaling is active in the pancreas mesenchyme , possibly mediated by bmps expressed within the mesenchyme . mrna ish , showing the expression of bmp4 and bmp7 at the level of the posterior stomach ( in a and c , arrows point at the dorsal aorta . arrowheads in a point to mesonephros , and arrowheads in c point to expression in the stomach mesenchyme ) and the pancreas ( b and d ) at hh . note that the mesenchymal expression of bmp4 and bmp7 in the pancreas mesenchyme ( arrows , b and d ) . ( a high - quality digital representation of this figure is available in the online issue . ) to investigate the requirements for bmp signaling in pancreas development , we applied two complementary approaches . we ectopically expressed the secreted bmp antagonist noggin or a dominant negative bmp receptor type 1 ( dnalk3 ) in the pancreas epithelium using in ovo electroporation of the endoderm ( 6,27 ) . we expected that the secreted antagonist noggin should block bmp signaling in the epithelium as well as in the mesenchyme , whereas dnalk3 should be autonomous to the epithelial cells expressing this construct . 1012 , and the introduced genes were thus expressed soon after the specification of the pancreas endoderm ( 17 ) . first we electroporated with a cmyc tagged noggin in the pcig5 vector which has an ires nuclear enhanced green fluorescent protein ( egfp ) sequence allowing direct monitoring of electroporated cells ( 6 ) . as expected , egfp was restricted to the epithelium after 32 h. however , cmyc immunoreactivity demonstrated that noggin was efficiently secreted and spread throughout the surrounding mesenchyme ( supplementary fig . we next assayed for psmad1,5,8 in the pancreas 24 and 48 h after electroporation ( fig . 3 ) . electroporation with a control plasmid did not change the pattern of endogenous psmad1,5,8 ( fig . 3b and f ) , but ectopic expression of noggin resulted in loss of mesenchymal psmad1,5,8 immunoreactivity around the electroporated area ( fig . we could still detect psmad1,5,8 in the mesenchyme at a distance from the electroporated area ( fig . we noted that the nkx6.1-positive pancreas epithelium appeared less developed than the corresponding controls after 48 h ( compare fig . ectopic expression of noggin in the chicken pancreas epithelium leads to loss of psmad1,5,8 immunoreactivity in the mesenchyme . as indicated , 24 h ( a  d ) and 48 h ( e  h ) after electroporation . note the loss of psmad1,5,8 in the pancreas mesenchyme around the noggin transfected area compared with controls . h : arrowheads point to psmad1,5,8 signals maintained at a distance from the electroporated area . ( a high - quality digital representation of this figure is available in the online issue . ) to assess pancreas morphogenesis , we performed whole - mount immunofluorescent staining of electroporated embryos at 24 , 48 , and 72 h. three - dimensional projections of confocal z - stacks were generated showing green fluorescent protein ( gfp ) , nkx6.1 , and glucagon / insulin ( fig . after electroporation with control plasmid or noggin , the dorsal and ventral pancreata were both formed normally , and endocrine cells were found exclusively in the dorsal pancreas ( fig . d ) . at 48 h , the pancreata electroporated with empty vector developed normally with primary branching , two distinct ventral pancreas buds ( 5 ) , and endocrine cells confined to the dorsal pancreas ( fig . instead the dorsal pancreas appeared as a tubular structure extending anteriorly along the stomach ( fig . 4 g and h ) . quantifying the amount of epithelial and endocrine areas showed that the total endocrine area was unchanged , but precocious endocrine cells could be observed in the ventral pancreas which was hypoplastic ( fig . 4 g and h , arrow ; supplementary table 2 ) . in some embryos , ectopic noggin expression in the pancreas results in poor epithelial branching , precocious endocrine differentiation . d : at 24 h after electroporation with control plasmid ( a and b ) and noggin ( c and d ) . projected images from z - stacks show correct specification of the dorsal and ventral pancreas under both conditions . b and d are identical to a and c , except that the gfp signal was removed to show the distribution of pancreatic cell types . e and f : forty - eight hours after electroporation with control plasmid ( e and f ) and noggin ( g and h ) . e and g : projected images from a z - stack . f and h : optical sections from the stack . in the control , primary branchpoint in the epithelium can be observed ( f , arrowheads ) and endocrine cells are restricted to the dorsal pancreas . the dorsal pancreas is completely unbranched after noggin electroporation and extends as a tubular structure anteriorly along the stomach ( arrowheads in g and h ) . numerous endocrine cells can be seen in the ventral pancreas ( arrow in g and h ) . l : seventy - two hours after electroporation with control plasmid ( i and j ) and noggin ( k and l ) . i and k : projected images from a z - stack . j and l : optical sections from the stack . branching is more pronounced in the controls after 72 h ( j , arrowheads ) , and endocrine cells a large cluster of pancreatic endocrine cells can be found detached from the pancreas in the stomach mesenchyme ( k and l , arrowhead ) . in the small remaining ventral pancreas , m : less frequently observed phenotype 48 h after noggin electroporation  the dorsal pancreas is almost entirely converted into a large endocrine cell mass . n : electroporation with dnalk3 has no effect on the normal pancreas development 48 h after electroporation . see table 1 for quantifications . ( a high - quality digital representation of this figure is available in the online issue . ) at 72 h after electroporation with control vector , endocrine cells were still confined to the dorsal pancreas and branching of the epithelium was prominent ( fig . 4i and j ) . conversely , the pancreata of noggin electroporated embryos were hypomorphic , and large endocrine clusters detached from the pancreas could be observed in the stomach mesenchyme ( fig . there was no epithelial branching and endocrine cells were found in the ventral pancreas ( fig . cell - autonomous inhibition of bmp signaling in the endoderm via electroporation with dnalk3 had no effect on pancreas development ( fig . 4n ) , suggesting that the effect elicited by noggin is through inhibition of bmp signaling in the mesenchyme . in neural tube electroporation experiments , dnalk3 , as well as noggin , was able to block smad1,5,8 phosphorylation in the dorsal neural tube . confirming that the dnalk3 construct worked as expected ( supplementary fig . 2c  h ) the pancreas of noggin electroporated embryos was further analyzed on sections 48 h after electroporation ( fig . the anterior extension of the pancreas contained nkx6.1 , glucagon , and insulin - positive cells ( fig . 5c ) , but no exocrine cells were found in noggin electroporated embryos ( fig . pancreatic cell types can be observed in the stomach mesenchyme and exocrine differentiation is reduced 48 h after electroporation with noggin . a and b are adjacent sections at the level of the stomach in a noggin electroporated embryo showing nkx6.1 mrna expression and insulin and glucagon protein expression , respectively . large clusters of pancreatic cells can be found in the stomach mesenchyme , and both glucagon and insulin are readily found ( a and b ) . c and d : sections of the pancreas of control ( c ) and noggin ( d ) electroprated embryos imunoflurescently labeled for amylase and showing gfp expression in electroporated cells . in d inset : the boxed area is without gfp . note the absence of amylase - positive cells in the noggin - electroporated pancreas . ( a high - quality digital representation of this figure is available in the online issue . ) the results presented so far indicate that bmp signaling is required in the mesenchyme and that the effect on the epithelium is indirect . this was difficult to demonstrate directly since electroporation in the chicken embryo does not permit cell - autonomous inhibition of bmp signaling in the mesenchyme . therefore , we took advantage of established protocols for in vitro development of mouse pancreas explants and lentiviral gene transduction ( 28 ) . we explanted e12.5 pancreata transduced with lentivirial vectors encoding either gfp , noggin , or dnalk3 . when whole pancreas explants were exposed to lentiviral vectors , exclusive infection of the mesenchyme cell autonomous bmp signaling inhibition in the mouse pancreas mesenchyme results in increased -cell differentiation . mouse pancreas organ cultures explanted and transduced at e12.5 ( a  k ) or 10.5 ( l  n ) with lentiviral constructs as indicated . a d  f : stereomicrographs of three different explants of each condition after 7 days in culture . g and h : explants transduced with a control egfp construct , immunflurescently stained for e - cadherin ( red ) and showing egfp with or without dapi nuclear counterstain . note how gfp expression is restricted to the mesenchyme 3 and 7 days after transduction . immunostained for amylase ( green ) , insulin ( red ) , and nuclei ( dapi , blue ) . n : explants transduced at e10.5 and cultured for 8 days , then stained for amylase and insulin with dapi nuclear counter stain . see table 2 for quantifications . ( a high - quality digital representation of this figure is available in the online issue . ) stereomicrographs of the explants cultured for 7 days showed that dnalk3 and noggin - transduced explants had a similar morphology and were of similar size as controls ( fig . f ) . costaining for e - cadherin and gfp on gfp - transduced explants confirmed the mesenchymal - specific expression after 3 and 7 days in culture ( fig . we quantified the number of exocrine cells and -cells after 7 days in culture ( fig . l , supplementary table 3 ) and found a 2.8-fold increase in the relative area of -cells in dnalk3-transduced explants compared with controls . this expansion was even more pronounced in noggin - transduced explants which showed a 3.6-fold increase in relative -cell area . in contrast , the relative exocrine area was diminished in dnalk3 and noggin - transduced explants compared with controls ( not statistically significant ) . there was no obvious branching defect in these studies , which could be a result of different timing of bmp inhibition compared with the studies performed in chicken . e10.5 gut tubes were dissected ; transduced with gfp , noggin , or dnalk3 ; and cultured for 4 and 8 days . after 4 days , the total explant volume was reduced by 5.5- and 1.8-fold when transduced with noggin and dnalk3 , respectively ( compared with control gfp transduction ; supplementary fig . noggin transduction resulted in a subtle change in the overall branching pattern , visualized by whole - mount mucin-1 staining , as main ducts were not well defined and appeared diffuse compared with controls ( supplementary fig . mesenchymal noggin expression increased the total number of insulin - positive cells by 3.8-fold , whereas the total number of amylase - positive cells were reduced by 1.6-fold ( fig . similarly , mesenchymal expression of dnalk3 increased the number of insulin - positive cells by 2.5-fold , demonstrating that cell autonomous bmp signal reception in the mesenchyme is required for a normal rate of endocrine cell differentiation to occur . we did observe a small but statistically significant effect on total exocrine cell numbers in noggin - transduced explants and in relative numbers of exocrine cells in both noggin and dnalk3 transduced ( supplementary table 3 ) . the effect of mesenchymal expression of dnalk3 on branching morphogenesis was ambiguous ; some explants exhibited a phenotype similar to the noggin - transduced explants , whereas some had a normal appearance ( not shown ) . since our results suggested that noggin inhibited bmp signaling in the mesenchyme , we next investigated mesenchymal gene expression patterns . pitx2 is normally expressed in the left side of the mesenchyme of vertebrate embryos and is involved in asymmetrical organ development ( 29,30 ) . bapx1 was recently demonstrated to be involved in the left - sided lateral growth of the e10.5 mouse dorsal pancreas because of a requirement for bapx1 in the formation of a specialized mesothelial structure , the splancnic mesodermal plate ( smp ) ( 31 ) . it is normally expressed in the left side of the mesenchyme , and it is required for the separation of the splenic and pancreatic mesenchyme and maintenance of the pancreas character in the distal part of the dorsal pancreas ( 31,32 ) . to evaluate the expression of pitx2 and bapx1 in relation to the chicken pancreas , we first performed mrna in situ hybridizations on serial sections for nkx6.1 and shh , together with pitx2 and bapx1 ( supplementary fig . 4 ) . we found that , as in the mouse , bapx1 was initially expressed bilaterally in the pancreas region ( supplementary fig . 4p ) , but from e3 strong expression was restricted to the pitx2-positive mesenchyme on the left side of the pancreas ( supplementary fig . bapx1 appeared to be symmetrically expressed at the level of the stomach ( supplementary fig . , we found that bapx1 was expressed left sided in the pancreas mesenchyme together with pitx2 after 48 h ( fig . 7c and e ) . in contrast , noggin - electroporated embryos displayed a duplication of bapx1 expression , whereas pitx2 remained left sided , suggesting that bapx1 and pitx2 expression is uncoupled at least under these conditions ( fig . these results are in agreement with a role for bmp signaling in development of the pancreas mesenchyme . since bapx1 is involved in the formation of the spm in the mouse embryo , we hypothesized that this structure could be affected in the bmp - inhibited embryos . we therefore stained for isl1 and laminin or nkx6.1/nkx2.2 with dapi nuclear counter stain to visualize this structure ( supplementary fig . , the spm was asymmetric with a condensation of the mesothelium on the left side at hh st . 5c and d ) , suggesting that downregulation of right - sided bapx1 expression is required for establishment of the asymmetric spm . electroporation with noggin results in persistence of a bapx1-positive pancreas mesenchyme on the right side of the pancreas 48 h after electroporation . a , c , e and b , d , f are serial sections from the same embryos , g  j are serial sections from other embryos . pitx2 and bapx1 are normally coexpressed on the left side of the pancreas in control embryos ( arrows , c and e ) . however , in noggin - electroporated embryos , bapx1 expression becomes symmetrically expressed on both sides of the pancreas ( arrows , f ) , whereas pitx2 remains left sided ( arrow , d ) . most of the unbranced dorsal pancreas in noggin electroporated embryos does not express hes1 ( g and h ) , but the duodenum maintains hes1 expression . the observation of precocious endocrine differentiation in noggin - electroporated embryos prompted us to analyze the expression of hes1 . we found that the unbranched tubular dorsal pancreas was hes1-negative 48 h after electroporation , suggesting that the excessive endocrine differentiation is coincident with downregulated hes1 expression ( fig . 7e and k ) . we therefore looked after 32 h of development and earlier ( supplementary fig . 6 and data not shown ) . most of the nkx6.1-positive dorsal pancreas epithelium had differentiated after 32 h and was hes1-negative , but the remaining nkx6.1-positive cells expressed normal levels of hes1 . at earlier time points when the pancreas was more normal , we could not detect a general downregulation of hes1 in the epithelium ( not shown ) . we can therefore not distinguish between loss of hes1 followed by differentiation or vice versa , likely because our ish protocol does not allow quantitative measurements of mrna expression levels . the bilateral expression of bapx1 in the mesenchyme of noggin electroporated embryos was notable . in chicken and mice , the right omphalomesenteric vein is normally found at this position , and the developing pancreas grows around this vessel ( 4,5 ) . gene expression changes in this structure might indicate that the normal intimately joined development of the pancreas and the vein was disturbed . to visualize the vasculature and the pancreas , we injected embryos with fluorescently - labeled lens culinaris agglutin , a lectin with high affinity to chicken endothelial cells ( 20 ) , and subsequently we stained for nkx6.1 by whole - mount immunohistochemistry ( fig . 8) . embryos perfused with a fluorescently - tagged lens culinaris agglutinin ( labeling the vascular endothelium ) and subjected to whole - mount immunostaing for nkx6.1 and gfp . a , b and e , f show projected z - stacks from lateral views at two different magnifications . c and g : projected images from transverse thick vibratome sections from the same embryos ( indicated by dashed lines in a and e ) . note how the dorsal aorta is well separated from the pancreas in the control and how the dorsal pancreas curls around the omphalomesenteric vein . in noggin - electroporated embryos , the omphalomesenteric vein is consistently found ventrally to the pancreas , and in some of the embryos , the paired dorsal aortas fail to fuse . the bisymmetrical mesenchyme is indicated by dashed lines in f. ( the contrast was temporarily enhanced in the image to allow the drawing based on a background signal . the mesenchyme is not readily observable at the published contrast setting ) . d and h : schematic drawing summarizing the relationship between the gut and pancreas ( green ) and the omphalomesenteric vein ( red ) . a , aorta ; dp , dorsal pancreas ; duo , duodenum ; dv , ductus venosus ; li , liver ; ov , omphalomesenteric vein ; vp , ventral pancreas . ( a high - quality digital representation of this figure is available in the online issue . ) at 3 days of development ( hh st . 19 ) , the paired omphalomesenteric veins run on each side of the gut tube and join anteriorly at the level of the liver in the ductus venosus ( 33 ) ( supplementary fig . the bilateral , ventral nkx6.1 domains are in close contact to these veins and the dorsal pancreas has grown asymmetrically over the right omphalomesenteric vein ( supplementary fig . the dorsal aorta is well separated from the pancreas by the pancreatic mesenchyme . during the next day , a series of anastomoses of the veins occur and most of the left omphalomesenteric vein regresses , leaving behind the right omphalomesenteric vein which curls around the gut at the level of the pancreas ( 5,33 ) ( supplementary fig . one of the ventral pancreata lie in close contact to the right omphalomesenteric vein ( supplementary fig . we analyzed the vascular development in control and noggin electroporated embryos 48 h after electroporation . 8c ) , and , as expected , the dorsal aorta was well separated from the dorsal pancreas ( fig . the omphalomesenteric vein was found on the ventral side of the pancreas and gut ( n = 6 ) and , in some embryos , the dorsal aortas failed to fuse and move dorsally ( 2 of 6 embryos ) . to assess the development of the microvasculature , we made flk1 ish on sectioned embryos . this revealed a normal microvasculature around the dorsal pancreas 32 h after electroporation when the omphalomesenteric vein is already displaced to the ventral side in bmp - inhibited embryos ( supplementary fig . bmp signaling is transduced by smad1 , 5 , and 8 which , upon signaling , become phosphorylated and accumulates in the nucleus where they act as transcription factors ( 23 ) . we used a phosphosmad1,5,8-specific antibody ( 24 ) to characterize endogenous bmp signaling in the embryonic chicken pancreas ( fig . 1 ) at hamburger and hamilton ( hh ) stages 12 , 18 , and 21 ( 25 ) . this corresponds to time points when the pancreas has just been specified in the endoderm , dorsal and ventral buds have formed , and -cells , -cells , and amylase - positive cells can be detected ( 6,17 ) . psmad1,5,8 stainings on e2 , e3 , and e4 ( hh st.12 , 18 , and 21 ) chicken embryos ( a  j ) , e9.5 mouse embryo ( k  l ) , and e11.5-dissected mouse gut ( m  n ) . images are either projections of confocal z - stacks of thick vibratome sections ( a  c ) or an optical section ( d ) . boxed area in b is shown in higher magnification in c and d. a : projected image from a z - stack showing psmad1,5,8 reactivity in the ventral forgut endoderm ( arrows ) and dorsal neural tube ( arrowhead ) . b : projected image at the presumptive pancreas level . note psmad1,5,8 reactivity in the lateral plate mesoderm and the underlying endoderm that appears yellow because of coexpression with foxa2 ( arrow ) . c and d : psmad1,5,8 reactivity can be observed in the endothelial cells of the dorsal aortas and in endoderm lateral to the nkx6.1 expression domain ( arrowhead ) . e and f : a section of an e3 pancreas stained as indicated ( shown with and without nkx6.1 ) . note the psmad1,5,8 reactivity in the mesenchyme and the absence of staining in the pancreas epithelium ( arrows point to the dorsal pancreas ) . g : composite of two images showing the dorsal and ventral pancreas and the liver ; psmad1,5,8 can be detected in the pancreas mesenchyme and in mesenchymal and epithelial compartments of the liver . h  j : another section from the same pancreas showing absence of psmad1,5,8 immunoreactivity in the nkx6.1-positive pancreatic epithelium and -cells ( recognizable by their morphology , arrowhead ) . k and l : optical sections from a z - stack of an e9.5 mouse stained in whole mount ; psmad1,5,8 can be detected in the dorsal aorta and intersomitic vessels and in mesenchymal cells in close contact with the ventral pancreas epithelium . a , aorta ; dp , dorsal pancreas ; duo , duodenum , v , right omphalomesenteric vein ; vp , ventral pancreas . ( a high - quality digital representation of this figure is available in the online issue . ) at hh st . 12 , embryos were whole - mount stained and 200-m transverse vibratome sections were analyzed ( fig . d ) and in the ventral part of the anterior foregut endoderm ( arrows , fig . 1a , ) as previously reported ( 24 ) . at the level of the 3rd to 8th somite pairs , where the prospective pancreatic endoderm is located ( 3,26 ) , psmad1,5,8 was detected in the lateral plate mesoderm and in the endoderm lateral to the nkx6.1 expression domain ( fig . 1 ) . we could also observe psmad1,5,8 in the endothelium of the paired dorsal aortas ( fig . 18 , psmad1,5,8 immunoreactivity was detected in the pancreas mesenchyme and at lower levels in the duodenal epithelium ( fig . one day later , we still observed a mesenchymal psmad1,5,8 signal ( fig . 1g j ) , but no signal was detected in the dorsal or ventral pancreatic epithelium . the liver epithelium had a strong signal with a sharp border of immunoreactivity in the bile duct ( fig . 1 g arrows ) . no psmad1,5,8 reactivity was detected in nkx6.1-positive cells , which at this stage comprise both undifferentiated epithelial cells and -cells ( 17 ) ( fig . the absence of labeling in -cells was confirmed by colabeling with an insulin antibody ( supplementary fig . 1 g and h ) . to evaluate the status of smad1,5,8 phosphorylation in the early mouse pancreas , we performed whole - mount staining at e9.5 and e11.5 for psmad1,5,8 combined with pdx1 ( fig . no psmad1,5,8 was detected in the pdx1-positive endoderm . at e11.5 , we observed psmad1,5,8 reactivity in the pancreas mesenchyme , but no reactivity in the pancreas epithelium ( fig we conclude that endogenous signaling leading to detectable levels of psmad1,5,8 in the early chicken and mouse pancreas is entirely restricted to the mesenchyme . bmp4 , -5 , and -7 mrna have been reported to be expressed in the embryonic mouse pancreas by rt - pcr ( 15 ) , and bmp4 and bmp7 have been localized to the pancreas epithelium ( 16 ) , but little is known about bmp expression during chicken pancreas development . 22 ) to characterize the expression patterns of bmp2 , bmp4 , bmp5 , and bmp7 ( fig . 2 and data not shown ) . we found that bmp4 and bmp7 are expressed in the chicken pancreas mesenchyme and excluded from the epithelium at the stages examined ( fig . we could not detect bmp2 or bmp5 in the pancreas at any stage ( data not shown ) . from these results we concluded that bmp signaling is active in the pancreas mesenchyme , possibly mediated by bmps expressed within the mesenchyme . mrna ish , showing the expression of bmp4 and bmp7 at the level of the posterior stomach ( in a and c , arrows point at the dorsal aorta . arrowheads in a point to mesonephros , and arrowheads in c point to expression in the stomach mesenchyme ) and the pancreas ( b and d ) at hh . note that the mesenchymal expression of bmp4 and bmp7 in the pancreas mesenchyme ( arrows , b and d ) . ( a high - quality digital representation of this figure is available in the online issue . ) to investigate the requirements for bmp signaling in pancreas development , we applied two complementary approaches . we ectopically expressed the secreted bmp antagonist noggin or a dominant negative bmp receptor type 1 ( dnalk3 ) in the pancreas epithelium using in ovo electroporation of the endoderm ( 6,27 ) . we expected that the secreted antagonist noggin should block bmp signaling in the epithelium as well as in the mesenchyme , whereas dnalk3 should be autonomous to the epithelial cells expressing this construct . 1012 , and the introduced genes were thus expressed soon after the specification of the pancreas endoderm ( 17 ) . first we electroporated with a cmyc tagged noggin in the pcig5 vector which has an ires nuclear enhanced green fluorescent protein ( egfp ) sequence allowing direct monitoring of electroporated cells ( 6 ) . as expected , egfp was restricted to the epithelium after 32 h. however , cmyc immunoreactivity demonstrated that noggin was efficiently secreted and spread throughout the surrounding mesenchyme ( supplementary fig . we next assayed for psmad1,5,8 in the pancreas 24 and 48 h after electroporation ( fig . 3 ) . electroporation with a control plasmid did not change the pattern of endogenous psmad1,5,8 ( fig . 3b and f ) , but ectopic expression of noggin resulted in loss of mesenchymal psmad1,5,8 immunoreactivity around the electroporated area ( fig . we could still detect psmad1,5,8 in the mesenchyme at a distance from the electroporated area ( fig . we noted that the nkx6.1-positive pancreas epithelium appeared less developed than the corresponding controls after 48 h ( compare fig . 3g ectopic expression of noggin in the chicken pancreas epithelium leads to loss of psmad1,5,8 immunoreactivity in the mesenchyme . sectioned pancreata immunolabeled as indicated , 24 h ( a  d ) and 48 h ( e  h ) after electroporation . note the loss of psmad1,5,8 in the pancreas mesenchyme around the noggin transfected area compared with controls . h : arrowheads point to psmad1,5,8 signals maintained at a distance from the electroporated area . ( a high - quality digital representation of this figure is available in the online issue . ) to assess pancreas morphogenesis , we performed whole - mount immunofluorescent staining of electroporated embryos at 24 , 48 , and 72 h. three - dimensional projections of confocal z - stacks were generated showing green fluorescent protein ( gfp ) , nkx6.1 , and glucagon / insulin ( fig . after electroporation with control plasmid or noggin , the dorsal and ventral pancreata were both formed normally , and endocrine cells were found exclusively in the dorsal pancreas ( fig . d ) . at 48 h , the pancreata electroporated with empty vector developed normally with primary branching , two distinct ventral pancreas buds ( 5 ) , and endocrine cells confined to the dorsal pancreas ( fig . 4e and f ) . instead the dorsal pancreas appeared as a tubular structure extending anteriorly along the stomach ( fig . 4 g and h ) . quantifying the amount of epithelial and endocrine areas showed that the total endocrine area was unchanged , but precocious endocrine cells could be observed in the ventral pancreas which was hypoplastic ( fig . 4 g and h , arrow ; supplementary table 2 ) . in some embryos , ectopic noggin expression in the pancreas results in poor epithelial branching , precocious endocrine differentiation . d : at 24 h after electroporation with control plasmid ( a and b ) and noggin ( c and d ) . projected images from z - stacks show correct specification of the dorsal and ventral pancreas under both conditions . b and d are identical to a and c , except that the gfp signal was removed to show the distribution of pancreatic cell types . e and f : forty - eight hours after electroporation with control plasmid ( e and f ) and noggin ( g and h ) . e and g : projected images from a z - stack . f and h : optical sections from the stack . in the control , primary branchpoint in the epithelium can be observed ( f , arrowheads ) and endocrine cells are restricted to the dorsal pancreas . the dorsal pancreas is completely unbranched after noggin electroporation and extends as a tubular structure anteriorly along the stomach ( arrowheads in g and h ) . numerous endocrine cells can be seen in the ventral pancreas ( arrow in g and h ) . l : seventy - two hours after electroporation with control plasmid ( i and j ) and noggin ( k and l ) . i and k : projected images from a z - stack . j and l : optical sections from the stack . branching is more pronounced in the controls after 72 h ( j , arrowheads ) , and endocrine cells are still confined to the dorsal pancreas . in noggin a large cluster of pancreatic endocrine cells can be found detached from the pancreas in the stomach mesenchyme ( k and l , arrowhead ) . in the small remaining ventral pancreas , m : less frequently observed phenotype 48 h after noggin electroporation  the dorsal pancreas is almost entirely converted into a large endocrine cell mass . n : electroporation with dnalk3 has no effect on the normal pancreas development 48 h after electroporation . see table 1 for quantifications . ( a high - quality digital representation of this figure is available in the online issue . ) at 72 h after electroporation with control vector , endocrine cells were still confined to the dorsal pancreas and branching of the epithelium was prominent ( fig . 4i and j ) . conversely , the pancreata of noggin electroporated embryos were hypomorphic , and large endocrine clusters detached from the pancreas could be observed in the stomach mesenchyme ( fig . there was no epithelial branching and endocrine cells were found in the ventral pancreas ( fig . cell - autonomous inhibition of bmp signaling in the endoderm via electroporation with dnalk3 had no effect on pancreas development ( fig . 4n ) , suggesting that the effect elicited by noggin is through inhibition of bmp signaling in the mesenchyme . in neural tube electroporation experiments , dnalk3 , as well as noggin , was able to block smad1,5,8 phosphorylation in the dorsal neural tube . confirming that the dnalk3 construct worked as expected ( supplementary fig . 2c  h ) . the pancreas of noggin electroporated embryos was further analyzed on sections 48 h after electroporation ( fig . the anterior extension of the pancreas contained nkx6.1 , glucagon , and insulin - positive cells ( fig . 5c ) , but no exocrine cells were found in noggin electroporated embryos ( fig . pancreatic cell types can be observed in the stomach mesenchyme and exocrine differentiation is reduced 48 h after electroporation with noggin . a and b are adjacent sections at the level of the stomach in a noggin electroporated embryo showing nkx6.1 mrna expression and insulin and glucagon protein expression , respectively . large clusters of pancreatic cells can be found in the stomach mesenchyme , and both glucagon and insulin are readily found ( a and b ) . c and d : sections of the pancreas of control ( c ) and noggin ( d ) electroprated embryos imunoflurescently labeled for amylase and showing gfp expression in electroporated cells . in d inset : the boxed area is without gfp . note the absence of amylase - positive cells in the noggin - electroporated pancreas . ( a high - quality digital representation of this figure is available in the online issue . ) the results presented so far indicate that bmp signaling is required in the mesenchyme and that the effect on the epithelium is indirect . this was difficult to demonstrate directly since electroporation in the chicken embryo does not permit cell - autonomous inhibition of bmp signaling in the mesenchyme . therefore , we took advantage of established protocols for in vitro development of mouse pancreas explants and lentiviral gene transduction ( 28 ) . we explanted e12.5 pancreata transduced with lentivirial vectors encoding either gfp , noggin , or dnalk3 . when whole pancreas explants were exposed to lentiviral vectors , exclusive infection of the mesenchyme cell autonomous bmp signaling inhibition in the mouse pancreas mesenchyme results in increased -cell differentiation . mouse pancreas organ cultures explanted and transduced at e12.5 ( a  k ) or 10.5 ( l  n ) with lentiviral constructs as indicated . a d  f : stereomicrographs of three different explants of each condition after 7 days in culture . g and h : explants transduced with a control egfp construct , immunflurescently stained for e - cadherin ( red ) and showing egfp with or without dapi nuclear counterstain . note how gfp expression is restricted to the mesenchyme 3 and 7 days after transduction . immunostained for amylase ( green ) , insulin ( red ) , and nuclei ( dapi , blue ) . n : explants transduced at e10.5 and cultured for 8 days , then stained for amylase and insulin with dapi nuclear counter stain . see table 2 for quantifications . ( a high - quality digital representation of this figure is available in the online issue . ) stereomicrographs of the explants cultured for 7 days showed that dnalk3 and noggin - transduced explants had a similar morphology and were of similar size as controls ( fig . f ) . costaining for e - cadherin and gfp on gfp - transduced explants confirmed the mesenchymal - specific expression after 3 and 7 days in culture ( fig . we quantified the number of exocrine cells and -cells after 7 days in culture ( fig . l , supplementary table 3 ) and found a 2.8-fold increase in the relative area of -cells in dnalk3-transduced explants compared with controls . this expansion was even more pronounced in noggin - transduced explants which showed a 3.6-fold increase in relative -cell area . in contrast , the relative exocrine area was diminished in dnalk3 and noggin - transduced explants compared with controls ( not statistically significant ) . there was no obvious branching defect in these studies , which could be a result of different timing of bmp inhibition compared with the studies performed in chicken . e10.5 gut tubes were dissected ; transduced with gfp , noggin , or dnalk3 ; and cultured for 4 and 8 days . after 4 days , the total explant volume was reduced by 5.5- and 1.8-fold when transduced with noggin and dnalk3 , respectively ( compared with control gfp transduction ; supplementary fig . noggin transduction resulted in a subtle change in the overall branching pattern , visualized by whole - mount mucin-1 staining , as main ducts were not well defined and appeared diffuse compared with controls ( supplementary fig . after 8 days in culture , mesenchymal noggin expression increased the total number of insulin - positive cells by 3.8-fold , whereas the total number of amylase - positive cells were reduced by 1.6-fold ( fig . similarly , mesenchymal expression of dnalk3 increased the number of insulin - positive cells by 2.5-fold , demonstrating that cell autonomous bmp signal reception in the mesenchyme is required for a normal rate of endocrine cell differentiation to occur . we did observe a small but statistically significant effect on total exocrine cell numbers in noggin - transduced explants and in relative numbers of exocrine cells in both noggin and dnalk3 transduced ( supplementary table 3 ) . the effect of mesenchymal expression of dnalk3 on branching morphogenesis was ambiguous ; some explants exhibited a phenotype similar to the noggin - transduced explants , whereas some had a normal appearance ( not shown ) . since our results suggested that noggin inhibited bmp signaling in the mesenchyme , we next investigated mesenchymal gene expression patterns . pitx2 is normally expressed in the left side of the mesenchyme of vertebrate embryos and is involved in asymmetrical organ development ( 29,30 ) . bapx1 was recently demonstrated to be involved in the left - sided lateral growth of the e10.5 mouse dorsal pancreas because of a requirement for bapx1 in the formation of a specialized mesothelial structure , the splancnic mesodermal plate ( smp ) ( 31 ) . it is normally expressed in the left side of the mesenchyme , and it is required for the separation of the splenic and pancreatic mesenchyme and maintenance of the pancreas character in the distal part of the dorsal pancreas ( 31,32 ) . to evaluate the expression of pitx2 and bapx1 in relation to the chicken pancreas , we first performed mrna in situ hybridizations on serial sections for nkx6.1 and shh , together with pitx2 and bapx1 ( supplementary fig . 4 ) . we found that , as in the mouse , bapx1 was initially expressed bilaterally in the pancreas region ( supplementary fig . 4p ) , but from e3 strong expression was restricted to the pitx2-positive mesenchyme on the left side of the pancreas ( supplementary fig . bapx1 appeared to be symmetrically expressed at the level of the stomach ( supplementary fig . 4h ) as in the mouse ( 31 ) . in control embryos , we found that bapx1 was expressed left sided in the pancreas mesenchyme together with pitx2 after 48 h ( fig . 7c and e ) . in contrast , noggin - electroporated embryos displayed a duplication of bapx1 expression , whereas pitx2 remained left sided , suggesting that bapx1 and pitx2 expression is uncoupled at least under these conditions ( fig . these results are in agreement with a role for bmp signaling in development of the pancreas mesenchyme . since bapx1 is involved in the formation of the spm in the mouse embryo , we hypothesized that this structure could be affected in the bmp - inhibited embryos . we therefore stained for isl1 and laminin or nkx6.1/nkx2.2 with dapi nuclear counter stain to visualize this structure ( supplementary fig . , the spm was asymmetric with a condensation of the mesothelium on the left side at hh st . 5c and d ) , suggesting that downregulation of right - sided bapx1 expression is required for establishment of the asymmetric spm . electroporation with noggin results in persistence of a bapx1-positive pancreas mesenchyme on the right side of the pancreas 48 h after electroporation . mrna in situ hybridization on electroporated embryos 48 h after electroporation . a , c , e and b , d , f are serial sections from the same embryos , g  j are serial sections from other embryos . pitx2 and bapx1 are normally coexpressed on the left side of the pancreas in control embryos ( arrows , c and e ) . however , in noggin - electroporated embryos , bapx1 expression becomes symmetrically expressed on both sides of the pancreas ( arrows , f ) , whereas pitx2 remains left sided ( arrow , d ) . most of the unbranced dorsal pancreas in noggin electroporated embryos does not express hes1 ( g and h ) , but the duodenum maintains hes1 expression . the observation of precocious endocrine differentiation in noggin - electroporated embryos prompted us to analyze the expression of hes1 . we found that the unbranched tubular dorsal pancreas was hes1-negative 48 h after electroporation , suggesting that the excessive endocrine differentiation is coincident with downregulated hes1 expression ( fig . 7e and k ) . most of the nkx6.1-positive dorsal pancreas epithelium had differentiated after 32 h and was hes1-negative , but the remaining nkx6.1-positive cells expressed normal levels of hes1 . at earlier time points when the pancreas was more normal , we could not detect a general downregulation of hes1 in the epithelium ( not shown ) . we can therefore not distinguish between loss of hes1 followed by differentiation or vice versa , likely because our ish protocol does not allow quantitative measurements of mrna expression levels . the bilateral expression of bapx1 in the mesenchyme of noggin electroporated embryos was notable . in chicken and mice , the right omphalomesenteric vein is normally found at this position , and the developing pancreas grows around this vessel ( 4,5 ) . gene expression changes in this structure might indicate that the normal intimately joined development of the pancreas and the vein was disturbed . to visualize the vasculature and the pancreas , we injected embryos with fluorescently - labeled lens culinaris agglutin , a lectin with high affinity to chicken endothelial cells ( 20 ) , and subsequently we stained for nkx6.1 by whole - mount immunohistochemistry ( fig . 8) . embryos perfused with a fluorescently - tagged lens culinaris agglutinin ( labeling the vascular endothelium ) and subjected to whole - mount immunostaing for nkx6.1 and gfp . a , b and e , f show projected z - stacks from lateral views at two different magnifications . c and g : projected images from transverse thick vibratome sections from the same embryos ( indicated by dashed lines in a and e ) . note how the dorsal aorta is well separated from the pancreas in the control and how the dorsal pancreas curls around the omphalomesenteric vein . in noggin - electroporated embryos , the omphalomesenteric vein is consistently found ventrally to the pancreas , and in some of the embryos , the paired dorsal aortas fail to fuse . the bisymmetrical mesenchyme is indicated by dashed lines in f. ( the contrast was temporarily enhanced in the image to allow the drawing based on a background signal . d and h : schematic drawing summarizing the relationship between the gut and pancreas ( green ) and the omphalomesenteric vein ( red ) . a , aorta ; dp , dorsal pancreas ; duo , duodenum ; dv , ductus venosus ; li , liver ; ov , omphalomesenteric vein ; vp , ventral pancreas . ( a high - quality digital representation of this figure is available in the online issue . ) at 3 days of development ( hh st . 19 ) , the paired omphalomesenteric veins run on each side of the gut tube and join anteriorly at the level of the liver in the ductus venosus ( 33 ) ( supplementary fig . the bilateral , ventral nkx6.1 domains are in close contact to these veins and the dorsal pancreas has grown asymmetrically over the right omphalomesenteric vein ( supplementary fig . the dorsal aorta is well separated from the pancreas by the pancreatic mesenchyme . during the next day , a series of anastomoses of the veins occur and most of the left omphalomesenteric vein regresses , leaving behind the right omphalomesenteric vein which curls around the gut at the level of the pancreas ( 5,33 ) ( supplementary fig . one of the ventral pancreata lie in close contact to the right omphalomesenteric vein ( supplementary fig . we analyzed the vascular development in control and noggin electroporated embryos 48 h after electroporation . 8c ) , and , as expected , the dorsal aorta was well separated from the dorsal pancreas ( fig . in noggin electroporated embryos , the vasculature developed abnormally ( fig . 8e  g ) . the omphalomesenteric vein was found on the ventral side of the pancreas and gut ( n = 6 ) and , in some embryos , the dorsal aortas failed to fuse and move dorsally ( 2 of 6 embryos ) . to assess the development of the microvasculature this revealed a normal microvasculature around the dorsal pancreas 32 h after electroporation when the omphalomesenteric vein is already displaced to the ventral side in bmp - inhibited embryos ( supplementary fig . development of the pancreas depends on interactions between the epithelium and the mesenchyme as recognized since the studies by golosow and grobstein ( 12 ) , but few endogenous signaling factors have been identified . here , we used chicken in ovo endoderm electroporation timed to express the pancreatic transgenes after specification of the pancreas . detection of psmad1,5,8 shows that early pancreatic mesenchyme receives a bmp signal , possibly through alk3 , which is expressed in the chicken pancreas mesenchyme ( 34 ) . since we find bmp expression in the mesenchyme , it is possible that an autocrine signaling mechanism is occurring . epithelial expression of the secreted bmp antagonist noggin resulted in the loss of psmad1,5,8 immunoreactivity in the adjacent mesenchyme and affected epithelial and mesenchymal development . the epithelium displayed reduced branching , anterior extension of the dorsal pancreas into the stomach mesenchyme , and precocious endocrine differentiation . inhibiting bmp signaling compromised angiogenic remodeling that positions the right omphalomesenteric vein adjacent to the pancreas . instead , we observed a persistence of mesenchymal bapx1 expression , a bilateral smp , and failure of the dorsal aortas to fuse in the most severely affected embryos . we can not distinguish a direct effect on the endothelial cells from an indirect effect acting through the mesenchyme . however , the microvasculature seems normal in bmp - inhibited embryos , suggesting that some aspects of angiogenesis occur normally and the defect appears limited to major vessels . notably , the mouse explants corroborate the findings in chick , arguing against involvement of the major vessels . additionally , mesenchymal isl1 expression , dependent on signals from blood vessels ( 35 ) , is maintained . the results presented herein appear to conflict with a recent report by goulley et al . bmp signaling was perturbed by the expression of noggin and bmp4 in the pancreas epithelium using the pdx1 promoter , with no profound effect on pancreatic development . we speculate that this could be due to different levels of expression since establishment of high - expressing transgenic mouse lines used in their studies would be prevented by a severe vascular or pancreatic phenotype caused by embryonic or perinatal lethality , and thus a selection for low expressers . a similar study expressing bmp6 under the control of the pdx1 promoter resulted in complete pancreas agenesis at a late gestational age , and these mice displayed a hypomorphic spleen , suggesting that the mesenchyme was also affected ( 15 ) . the present study suggests that there is no epithelial bmp signaling in the pancreas at the investigated stages , and that the effect on epithelial development is secondary to inhibition of bmp signaling in the mesenchyme . this is supported by the observation that cell autonomous bmp - signaling inhibition in the mouse pancreas mesenchyme leads to an increase in endocrine differentiation concomitant with a decrease in exocrine development . it is possible that the effect of bmp inhibition on the mesenchyme can be subdivided into an inhibition of an autocrine mesenchymal signal and an associated effect on vascular remodelling . bmp signaling in the mesenchyme may be required for the mesenchyme to express a secondary factor acting on the epithelium . fgf10 is one candidate because mesenchymal fgf10 is required for epithelial branching and progenitor cell proliferation ( 911 ) . however , there is little fgf10 expression left at e12.5 , the time of explanting in some of the organ culture experiments , suggesting that other factors could be involved .