BACKGROUND
branching morphogenesis is a process that allows the formation of a branched network of tubes, as exemplified by the airways of the lung or the excretory ducts of the pancreas and salivary glands  <cit> .
during branching morphogenesis, the epithelial cells interact with the surrounding mesenchyme and organize into polarized monolayers with their apical pole facing the tube lumen  <cit> .
how this process takes place and is regulated in exocrine tissues such as the pancreas and salivary glands remains poorly understood.
in the mouse, the pancreas originates from a pre-patterned endodermal epithelium located in a caudal region of the foregut that is to become the duodenum.
between embryonic days   <dig>  and e <dig> , two outgrowths develop from the dorsal and ventral sides of this endodermal region, and form epithelial buds surrounded by mesenchyme.
from e <dig> -e <dig>  onwards, the pancreatic bud cells proliferate, differentiate and undergo extensive morphogenesis to generate ductal structures called primitive ducts.
the latter then expand, and give rise to the endocrine islets of langerhans and to a branched ductal network that drains the secretions of the exocrine acini  <cit> .
the submandibular glands  also derive from the foregut endoderm.
their development starts around e <dig>  by formation of two epithelial thickenings beneath the tongue.
these thickenings protrude into the underlying mesenchyme.
around e <dig> , small clefts appear at the periphery of the budding epithelial mass, and after continuous proliferation and repetitive clefting, a tree-like network of ducts whose branches end in acini is generated  <cit> .
regulation of epithelial morphogenesis in the pancreas and smg is controlled by the surrounding mesenchyme  <cit> .
moreover, gene inactivation studies and ex vivo culture experiments have identified several signaling molecules that regulate smg branching morphogenesis  <cit> .
in the developing pancreas, gene inactivation studies inhibiting fgf <dig>  egf, or rbpj expression revealed impaired branching morphogenesis.
however, these studies focused on the role of the signaling molecules on pancreatic cell differentiation and not on the mechanisms of branching  <cit> .
stromal cell-derived factor- <dig>  is a secreted protein of the α-chemokine family, and a potent chemoattractant for many cell types  <cit> .
whereas sdf- <dig> is the sole ligand for the chemokine cxc-motif receptor  <dig> , cxcr <dig> can bind sdf- <dig> and cxcl11/i-tac  <cit> .
sdf <dig> and cxcr <dig> knockout mice die perinatally and display profound defects in the hematopoietic and nervous system  <cit> , whereas cxcr <dig> knockout embryos die at birth due to defects in heart formation  <cit> .
no role has been ascribed to sdf-1/cxcr <dig> signaling in the smg.
in contrast, two functions for sdf- <dig> signaling in adult pancreas have been proposed.
one day before birth, when pancreatic cells still differentiate and extensive islet neogenesis occurs, cxcr <dig> is expressed in endocrine cells and in some ductal cells, whereas sdf- <dig> is only found in endocrine cells  <cit> .
the same expression pattern persists in adult pancreas.
using a genetic model of endocrine pancreas regeneration , it was proposed that sdf- <dig> is involved in endocrine cell renewal  <cit> .
more recently, it has been shown that sdf- <dig> promotes β-cell survival, via activation of akt, in adult mouse islets  <cit> .
however, it is not known if sdf-1/cxcr <dig> signaling plays a role in early pancreas development.
in this paper we show that pancreatic epithelial morphogenesis occurs according to a sequence of two opposite epithelial transitions.
during the first transition the monolayer of endodermal cells lining the primitive gut gives rise to two pancreatic buds, each composed of a mass of non polarized epithelial cells.
during the second transition, this mass reorganizes into branched polarized epithelial monolayers that further differentiate into ducts and acini.
similar sequential transitions were described earlier in the smg  <cit> .
in the pancreas and smg, sdf <dig> was expressed by the mesenchyme and cxcr <dig> by the epithelial cells.
using embryonic explants of pancreas and smg, and mice deficient in sdf- <dig> signaling, we uncovered a new role for sdf- <dig> in branching morphogenesis.
RESULTS
primitive duct morphogenesis and cell polarization during pancreas development
to obtain a better insight into the regulation of branching morphogenesis in developing pancreas, we first analyzed primitive duct morphogenesis and epithelial cell polarization.
we visualized epithelial cells , their apical pole lining the lumen , and the basal lamina  separating the epithelial cells from the mesenchyme on sections from e <dig>  to e <dig>  pancreas .
at e <dig>  and e <dig> , the pancreatic buds had a globular shape, and most epithelial cells were not polarized, as they were not lined by laminin  and did not show mucin- <dig> .
at e <dig> , the shape of the pancreas became irregular.
lumina of primitive ducts, still surrounded by several layers of epithelial cells became detectable from e <dig>  onwards.
the pancreas then progressively gave rise to the primitive ductal network, composed of a polarized and monolayered epithelium surrounded by mesenchyme.
at e <dig> , all epithelial cells were polarized and showed apical, lateral and basal domains .
very few apoptotic cells were detected during these stages, and they were exclusively localized in the non-epithelial compartment .
we concluded that the formation of the primitive pancreatic ductal network occurs by remodeling of a globular mass of epithelial cells, rather than from iterative branching of a monolayer.
we then further characterized the morphology and the cells of the e <dig>  pancreatic bud.
in addition to the central duct lumen , intense mucin- <dig> stainings also appeared as dots at the periphery of the pancreatic buds .
these stainings corresponded to small developing lumina, called here secondary lumina.
three-dimensional confocal reconstructions were generated to determine the spatial relationship between these secondary lumina and the central duct lumen.
no connection between the central and the peripheral lumina were detected, indicating that the latter are blind and independent from the central lumen .
furthermore, a section through the e <dig>  pancreatic bud stained for e-cadherin and mucin- <dig> expression showed that the cells of the outer layer are morphologically different from internal cells .
the outer cells had a cylindrical shape and a basal nucleus.
their apical pole faced the peripheral lumina, as shown by mucin- <dig> staining .
in addition, the localization of two intracellular markers in this outer epithelial layer was typical of polarized cells .
the golgi marker gm- <dig> was found on the apical side of the nucleus, and the microtubule-organizing center  detected by pericentrin staining was located in the subapical region of the cells.
in contrast, except for the cells that lined the central duct lumen, the internal cells of the pancreatic bud showed no mucin- <dig> expression and the localization of the golgi and mtoc was random.
altogether, these data indicated that the pancreatic bud is a compact epithelial mass, in which the cells are not polarized, except for the outer cells which all have basal and lateral membrane domains; some cells also posses an apical domain.
to characterize the acquisition of cell polarity during the transition of a globular pancreatic bud to the monolayered epithelium lining the primitive ducts, we analyzed the formation of tight junctions.
the latter play a role in cell adhesion and in the establishment and maintenance of apico-basal polarity  <cit> .
to this end, we studied the localization of the tight junction-specific protein zo- <dig> from e <dig>  to e <dig>  .
despite the continuous presence of adherens junctions  between pancreatic epithelial cells, strong zo- <dig> staining was not detected in the pancreas before e <dig> .
from that stage on, zo- <dig> was progressively seen in cells facing a lumen .
therefore, zo- <dig> localization at the tight junctions was subsequent to the onset of polarization of peripheral cells in the e <dig>  pancreatic bud and was concomitant with the remodeling of the epithelial bud into a monolayered epithelium lining the primitive duct lumina.
a similar epithelial transition, from a mass to monolayers, also occurs during submandibular gland  morphogenesis  <cit> .
as shown in figure 3b, zo- <dig> staining was not visible in the early stage of smg morphogenesis and only became visible after remodeling of the epithelial mass into monolayers lining a lumen .
high magnification pictures of epithelial cell monolayers clearly demonstrated that zo- <dig> separates the apical domain  from the lateral domain .
sdf- <dig> and its receptor cxcr <dig> are expressed in developing pancreas and smg
the mesenchyme is a source of morphogenesis-inducing signals in the pancreas  <cit>  and in the smg  <cit> .
therefore, we looked for mesenchymal factors that could regulate epithelial morphogenesis in these organs and considered sdf- <dig> as a candidate.
indeed, this chemokine and its g protein-coupled receptor cxcr <dig> have been proposed to control cell movement in many developing organs  <cit> , including the endoderm during zebrafish gastrulation  <cit> .
at the late stages of pancreas development, this signaling pathway is involved in pancreatic cell migration and β-cell survival  <cit> .
we first investigated the expression pattern of sdf- <dig> and cxcr <dig> in the pancreas by performing in situ hybridization experiments.
we focused on embryos at e <dig> , when epithelial cells are organized in a globular mass, and e <dig> , when remodeling has been initiated.
we found that sdf <dig> is mainly expressed in the mesenchyme, with lower staining intensity in the epithelium; cxcr <dig> was found in epithelial cells, as early as e <dig>  .
sdf <dig> and cxcr <dig> expression was also found in the vasculature .
this expression pattern was maintained up to e <dig> , a stage at which sdf <dig> expression in the central most region of the epithelium became intense .
in the developing smg, the expression of sdf <dig> and cxcr <dig> was comparable to that in the pancreas .
from e <dig>  to e <dig> : sdf <dig> was expressed in the mesenchyme and cxcr <dig> in the epithelium.
moreover, cxcr <dig>  the second receptor for sdf- <dig>  was observed by immunolocalization in endothelial structures of the smg , but not of the pancreas .
from this set of expression data, we concluded that the spatial and temporal expression pattern of sdf- <dig> and cxcr <dig> in the pancreas and smg suggests a potential role for sdf- <dig> signaling in their development.
sdf- <dig> signaling controls pancreatic branching morphogenesis
to address the role of sdf- <dig> signaling in epithelial morphogenesis, we resorted to explants of e <dig>  pancreas cultured on filters.
pancreatic cultures have only been validated for analyzing pancreatic cell differentiation when investigating the role of extracellular factors  <cit> .
therefore, we first validated the pancreatic explants as a model for studying branching epithelial morphogenesis.
at the start of the culture  the explants presented as an epithelial cell mass surrounded by mesenchyme, showing early signs of branching and small, independent, lumina.
after  <dig> to  <dig> days of culture, the epithelial mass was remodeled into tubulo-acinar epithelial monolayers imbricated within the mesenchyme, as described in vivo .
most epithelial cells had acquired polarity with the apical pole facing a well-defined lumen, as revealed by mucin- <dig> staining, and the basal pole facing the mesenchyme .
we concluded that cultured pancreatic explants provide a model to analyze the formation of the primitive ductal network.
to investigate if sdf- <dig> controls epithelial morphogenesis in developing pancreas, we performed gain- and loss-of-function experiments by treating cultured explants with exogenous recombinant sdf- <dig> and with a specific pharmacological inhibitor of cxcr <dig>   <cit> .
pancreatic explants cultured for  <dig> days were analyzed by immunofluorescence with antibodies against e-cadherin and mucin- <dig>  no effect of exogenous sdf- <dig> on epithelial organization was observed as compared to control explants .
the absence of effect of exogenous sdf- <dig> is likely to result from the presence of endogenous sdf- <dig>  whose activity cannot be amplified.
in contrast, we found that upon amd <dig> treatment, morphogenesis was impaired .
epithelial monolayers were not formed and the epithelial cells remained clustered.
the mesenchyme was not imbricated with the epithelium and remained at the periphery of the explants.
the lumina, as revealed by mucin- <dig> staining, were smaller and often surrounded by multiple layers of epithelial cells.
identical results were obtained with explants from e <dig>  embryos , and the effect of amd <dig> was dose-dependent, since milder anomalies were observed at  <dig> μm than at  <dig> μm.
thus, at the end of the culture, the amd3100-treated explants resembled the e <dig>  pancreas before the onset of remodeling into a monolayered epithelium .
amd <dig> treatment did not change the proliferation or the apoptosis index  and this was supported by the normal size of these explants.
immunostaining with antibodies against insulin, glucagon or carboxypeptidase-a revealed normal differentiation in the explants treated with amd <dig> .
from these experiments we concluded that sdf-1/cxcr <dig> signaling is potentially required for the remodeling of the epithelial cell mass into primitive pancreatic ducts lined by a monolayered epithelium.
sdf- <dig> signaling controls smg branching morphogenesis
to investigate if sdf- <dig> modulates branching morphogenesis of other exocrine glands, we addressed the role of sdf- <dig> signaling in smg epithelial morphogenesis.
as shown above, this organ develops according to the same two-stage epithelial transition as in the pancreas  <cit> .
we resorted to cultures of e <dig>  embryonic explants, as described earlier by others  <cit> .
the smg epithelial bud surrounded by its mesenchyme was excised and cultured on filter for up to  <dig> days in the presence of sdf- <dig> and sdf- <dig> antagonists.
similarly to the pancreas, gain-of-function experiments with exogenous sdf- <dig> did not affect smg branching .
however, upon amd <dig> treatment, morphogenesis of the smg was impaired .
the size of the glands was smaller and the number of epithelial buds was reduced, as compared to control glands, indicating that sdf- <dig> signaling is required for branching.
quantification of the number of buds in amd3100-treated explants revealed a reduction in the budding index , as early as one day after the onset of the treatment, and culminating at 80% reduction after  <dig> days, as compared to control cultures .
as cxcr <dig> was also expressed in the blood vessels surrounding the smg , the sdf- <dig> antagonist ccx <dig>  which prevents sdf- <dig> from binding to cxcr <dig>  was also tested.
as shown in figure 6a, ccx <dig> inhibited smg morphogenesis by reducing the number of epithelial buds.
this was confirmed by a 60% reduction in the budding index as compared to controls after  <dig> days .
these experiments suggested a role for sdf- <dig> signaling via cxcr <dig> and cxcr <dig> in the branching morphogenesis of the smg.
we further investigated the morphogenic defect observed by the addition of amd <dig> to the culture.
as sdf- <dig> signaling can control both cell survival and cell proliferation in different cell types, blocking sdf- <dig> binding to cxcr <dig> with amd <dig> could explain the reduced size of the glands.
therefore, we performed immunodetection of phosphohistone h <dig>  a marker of mitosis, and of activated caspase  <dig>  a marker of cell apoptosis, in control, amd3100- and ccx733-treated explants.
quantification of these stainings revealed that proliferation was not affected when sdf- <dig> signaling was abrogated .
however, apoptosis showed a three-fold increase in the epithelial buds of amd3100-treated explants .
this observation could explain the reduced size of the smg epithelium.
we then assessed whether the increase in apoptosis was the cause of the branching defect.
for this, we cultured explants in the presence of amd <dig> alone or in combination with a general inhibitor of caspases.
apoptosis was effectively prevented as revealed by the near complete absence of activated caspase  <dig> staining on sections from caspase inhibitor-treated explants .
however, in the explants co-cultured with amd <dig> and the caspases inhibitor, the branching defect was still observed , indicating that the reduced branching activity is not a consequence of increased apoptosis.
since sdf- <dig> did not affect cell survival in developing pancreas  but did control branching morphogenesis of the pancreas, we conclude that sdf- <dig> has differential effects on organ development, but that it has common effects on epithelial branching.
genetic deficiency of cxcr <dig> or sdf- <dig> transiently affects epithelial morphogenesis
to verify in vivo the relevance of our ex vivo observations, we analyzed the morphogenesis of embryonic pancreas in sdf <dig> knockout mice.
at e <dig>  the ventral pancreas in sdf <dig> knockouts showed less branching and had a more globular shape as compared to controls, confirming the role of sdf- <dig> in branching morphogenesis .
the degree of branching was evaluated by counting the number of buds and clefts in control and knockout ventral pancreas.
this quantification revealed a two-fold decrease in the number of buds in the absence of sdf- <dig> .
however, the dorsal pancreas did not show obvious branching defects, but was more elongated than in controls and it adopted a hammerhead-like structure while the controls were more rounded.
the branching defect was transient since e <dig> -e <dig> sdf1-/- embryos did not show deficient morphogenesis .
since the consequences of sdf <dig> ablation in vivo were rather modest as compared to the effects of sdf <dig> inhibition in vitro, we looked at cxcr4-/- pancreas.
at e <dig>  and e <dig> , the dorsal and ventral pancreata were not significantly different in cxcr4-/- embryos and in wild-type animals .
however, when cultured on filters cxcr4-/- pancreatic explants showed a strong morphogenic deficiency, similar to that seen when wild-type explants were cultured in the presence of amd <dig>  a sdf- <dig> antagonist .
this similarity proves that amd <dig> specifically targets and inhibits sdf- <dig> signaling via the cxcr <dig> receptor.
taken together, these data indicated that sdf- <dig> controls pancreas morphogenesis in vivo, but also suggest that compensatory effects operating in vivo mask part of the role of sdf- <dig>  we also analyzed the development of smg in cxcr4-/- and sdf1-/- embryos, but the smg phenotypes appeared normal, both in vitro and in vivo.
we concluded that no genetic evidence for a role of sdf- <dig> signaling in smg branching could be collected.
however, the existence of compensatory mechanisms for genetic deficiencies in the pancreas suggests that compensation may also occur in the smg.
discussion
in this paper we show that the primitive pancreatic ductal network, which is lined by a monolayered and polarized epithelium, forms by remodeling of a globular mass of non polarized epithelial cells.
sdf- <dig> is expressed in the mesenchyme surrounding the epithelium and its receptor cxcr <dig> is found in the epithelial cells of the pancreas.
sdf- <dig> signaling promotes branching morphogenesis of the pancreas in vitro and in vivo.
we also provide in vitro evidence that smg morphogenesis is controlled by sdf- <dig>
previous models depicting pancreatic duct morphogenesis in mammals suggest that it results from the branching of a polarized epithelium  <cit> .
we show here that formation of the primitive ductal network is not initiated by iterative branching of a tubular structure extending from the gut .
the pancreatic bud at e <dig> -e <dig>  consists of a globular mass of epithelial cells with a central duct lumen.
this lumen, connected to the gut tube lumen, forms extensions within the pancreatic bud, and is surrounded by several layers of cells.
these cells are linked by adherens junctions , but lack tight junctions .
at this stage, we observe that some cells at the periphery of the mass show apico-basal polarity and delineate blind lumina .
based on these observations, we propose that primitive duct branches arise from extensions of the central gut-derived duct that fuse with secondary lumina that appear at e <dig> .
interestingly, this process bears resemblance with lumen development in the zebrafish gut tube and exocrine pancreas.
indeed, in these organs the lumen arises by fusion of multiple lumina  <cit> .
after the formation of primitive ducts, further branching and expansion of the pancreatic ducts occur by iterative lateral branching, as illustrated by puri and hebrok  <cit> .
this is also similar to the development of the mouse smg, where the epithelial cells first form a mass that becomes remodeled into a monolayered epithelium.
like in the pancreas, epithelial cells in the smg express e-cadherin and β-catenin.
zo- <dig> is only detectable when cells are lining a lumen  <cit> .
the time-course and 3-d analysis of embryonic pancreas development indicated that an initial phase of polarization occurs in cells of the outermost layer of the pancreatic bud at e <dig> .
this precedes remodeling of the bud, suggesting that, at the onset of pancreas development, remodeling may be triggered by the acquisition of polarity and not the reverse.
beyond this stage, acquisition of apico-basal polarity and primitive duct formation cannot be dissociated morphologically but must be orchestrated by several regulators acting on distinct aspects.
indeed, in our culture explants, blocking sdf- <dig> signaling did not prevent epithelial cells in the mass to acquire apical characteristics such as assembly of tight junction complexes and primary cilia .
nevertheless, this polarization program could not go to completion, as the cells remained clustered and most of them did not acquire a basal domain.
during the remodeling of the pancreatic globular mass, the epithelium is in contact with the mesenchyme.
the latter tissue has been proposed to provide signals that control epithelial proliferation, differentiation and morphogenesis.
we have identified here sdf-1/cxcr <dig> signaling as a potential regulator of branching morphogenesis.
indeed, sdf- <dig> and its receptor, cxcr <dig>  are expressed in embryonic pancreas in a complementary fashion.
when epithelial cells are still organized as a non polarized mass, sdf- <dig> is expressed in the mesenchyme whereas cxcr <dig> is localized in the epithelium.
moreover, when sdf- <dig> signaling is inhibited by pharmacological means or by cxcr <dig> genetic deficiency, pancreas morphogenesis is perturbed.
these data are consistent with a model in which the mesenchymally-produced sdf- <dig> promotes epithelial remodeling and thus primitive duct morphogenesis.
while this work was under review, ueland and collaborators demonstrated that sdf- <dig> controls epithelial kidney morphogenesis in vitro  <cit> .
our data in the pancreas and smg, together with those of ueland et al.
demonstrate the role of sdf- <dig> in epithelial branching morphogenesis.
the molecular nature of the branching defects does not involve a change in the proliferation or apoptosis indexes , but may be explained by a change in the migration potential of epithelial cells  <cit> .
there is no perfect overlap between the results obtained with the pharmacological inhibition and with genetic inactivation of sdf- <dig> signaling in the pancreas.
pharmacological inhibition of sdf- <dig> signaling in vitro severely repressed branching morphogenesis, but genetic deficiency in sdf1-/- pancreas showed more modest effects in vivo.
also, the effect of cxcr <dig> gene ablation only led to abnormal pancreas morphogenesis when the organ was cultured in vitro.
we suggest that compensatory pathways may be active in vivo but not in vitro.
in our culture system, endothelial cells are present and organized around the epithelium like in vivo, the main difference being that there is no blood circulation in the endothelial network in vitro.
we suggest that signal coming directly or indirectly from the circulation could participate in the control of epithelial branching morphogenesis of the pancreas and smg.
such a model would further extend the role of the vasculature in pancreas development, since others previously showed that blood flow through the aorta is required for dorsal pancreas budding from the endoderm  <cit> .
in developing smg, cxcr <dig> is expressed in the epithelium and sdf- <dig> in the mesenchyme, like in the pancreas.
this suggests that sdf- <dig> controls branching morphogenesis by a direct mechanism on the epithelium.
importantly, cxcr <dig> is detected in endothelial cells of the smg, but not in the pancreas.
in smg explants, we observed a branching defect when sdf- <dig> binding to cxcr <dig> was inhibited by ccx <dig>  this suggests that sdf- <dig> can also indirectly control epithelial branching morphogenesis, via the endothelium.
activation of cxcr <dig> in the endothelium may induce the production of an unidentified factor which in turn would signal to the epithelium and act in parallel to or amplify the direct sdf- <dig> effects on epithelial morphogenesis.
in the smg we also noticed a difference between the effects of pharmacological inhibition of sdf- <dig> and genetic deficiencies induced by inactivation of the sdf <dig> or cxcr <dig> genes.
in vitro and in vivo development of cultured cxcr4-/- and sdf1-/- smg was normal, unlike development of amd3100- or ccx733-treated explants.
this suggests the existence of other ligands that could bind cxcr <dig>  or alternative signaling pathways with which sdf-1/cxcr4/ <dig> might interact in this organ.
it has been reported that migrating muscle progenitors cells reach the anlage of the tongue in cxcr4-/- or gab1-/-, but not in the double-mutant mice.
a crosstalk between g-protein-coupled receptors and tyrosine kinase receptors may contribute to compensatory mechanisms  <cit> .
finally, when sdf- <dig> signaling was blocked, the size of the epithelium was reduced in the smg but not in the pancreas.
although sdf- <dig> is important for survival of insulin-producing β-cells  <cit> , we did not observe any effect of sdf- <dig> signaling on cell survival early in pancreas development, even when e <dig>  explants were cultured in the absence of serum .
this indicates that the survival signals are different in the two organs.
a role for sdf- <dig> in cell survival has already been described in several systems  <cit> .
we excluded the possibility that the role of sdf- <dig> in cell survival in smg coincided with its role in branching morphogenesis, thereby indicating that sdf- <dig> plays distinct roles in the smg.
CONCLUSIONS
in conclusion, the present work shows that the formation of primitive ducts in the pancreas depends on remodeling of a globular mass of epithelial cells, as described earlier in smg.
our data also identify sdf- <dig> as a factor that may promote the transition from a mass of epithelial cells towards monolayers in the pancreas and smg.
understanding the mechanisms of such epithelial transition is relevant to carcinogenesis, which is associated with loss of epithelial polarity and formation of a mass of cells that fills the ductal lumen.

 branching morphogenesis is a process that allows the formation of a branched network of tubes, as exemplified by the airways of the lung or the excretory ducts of the pancreas and salivary glands. during branching morphogenesis, the epithelial cells interact with the surrounding mesenchyme and organize into polarized monolayers with their apical pole facing the tube lumen. how this process takes place and is regulated in exocrine tissues such as the pancreas and salivary glands remains poorly understood. in the mouse, the pancreas originates from a pre-patterned endodermal epithelium located in a caudal region of the foregut that is to become the duodenum. between embryonic days and e, two outgrowths develop from the dorsal and ventral sides of this endodermal region, and form epithelial buds surrounded by mesenchyme. from e -e onwards, the pancreatic bud cells proliferate, differentiate and undergo extensive morphogenesis to generate ductal structures called primitive ducts. the latter then expand, and give rise to the endocrine islets of langerhans and to a branched ductal network that drains the secretions of the exocrine acini. the submandibular glands also derive from the foregut endoderm. their development starts around e by formation of two epithelial thickenings beneath the tongue. these thickenings protrude into the underlying mesenchyme. around e, small clefts appear at the periphery of the budding epithelial mass, and after continuous proliferation and repetitive clefting, a tree-like network of ducts whose branches end in acini is generated. regulation of epithelial morphogenesis in the pancreas and smg is controlled by the surrounding mesenchyme. moreover, gene inactivation studies and ex vivo culture experiments have identified several signaling molecules that regulate smg branching morphogenesis. in the developing pancreas, gene inactivation studies inhibiting fgf egf, or rbpj expression revealed impaired branching morphogenesis. however, these studies focused on the role of the signaling molecules on pancreatic cell differentiation and not on the mechanisms of branching. stromal cell-derived factor- is a secreted protein of the α-chemokine family, and a potent chemoattractant for many cell types. whereas sdf- is the sole ligand for the chemokine cxc-motif receptor, cxcr can bind sdf- and cxcl11/i-tac. sdf and cxcr knockout mice die perinatally and display profound defects in the hematopoietic and nervous system, whereas cxcr knockout embryos die at birth due to defects in heart formation. no role has been ascribed to sdf-1/cxcr signaling in the smg. in contrast, two functions for sdf- signaling in adult pancreas have been proposed. one day before birth, when pancreatic cells still differentiate and extensive islet neogenesis occurs, cxcr is expressed in endocrine cells and in some ductal cells, whereas sdf- is only found in endocrine cells. the same expression pattern persists in adult pancreas. using a genetic model of endocrine pancreas regeneration, it was proposed that sdf- is involved in endocrine cell renewal. more recently, it has been shown that sdf- promotes β-cell survival, via activation of akt, in adult mouse islets. however, it is not known if sdf-1/cxcr signaling plays a role in early pancreas development. in this paper we show that pancreatic epithelial morphogenesis occurs according to a sequence of two opposite epithelial transitions. during the first transition the monolayer of endodermal cells lining the primitive gut gives rise to two pancreatic buds, each composed of a mass of non polarized epithelial cells. during the second transition, this mass reorganizes into branched polarized epithelial monolayers that further differentiate into ducts and acini. similar sequential transitions were described earlier in the smg. in the pancreas and smg, sdf was expressed by the mesenchyme and cxcr by the epithelial cells. using embryonic explants of pancreas and smg, and mice deficient in sdf- signaling, we uncovered a new role for sdf- in branching morphogenesis.  primitive duct morphogenesis and cell polarization during pancreas development to obtain a better insight into the regulation of branching morphogenesis in developing pancreas, we first analyzed primitive duct morphogenesis and epithelial cell polarization. we visualized epithelial cells, their apical pole lining the lumen, and the basal lamina separating the epithelial cells from the mesenchyme on sections from e to e pancreas. at e and e, the pancreatic buds had a globular shape, and most epithelial cells were not polarized, as they were not lined by laminin and did not show mucin-. at e, the shape of the pancreas became irregular. lumina of primitive ducts, still surrounded by several layers of epithelial cells became detectable from e onwards. the pancreas then progressively gave rise to the primitive ductal network, composed of a polarized and monolayered epithelium surrounded by mesenchyme. at e, all epithelial cells were polarized and showed apical, lateral and basal domains. very few apoptotic cells were detected during these stages, and they were exclusively localized in the non-epithelial compartment. we concluded that the formation of the primitive pancreatic ductal network occurs by remodeling of a globular mass of epithelial cells, rather than from iterative branching of a monolayer. we then further characterized the morphology and the cells of the e pancreatic bud. in addition to the central duct lumen, intense mucin- stainings also appeared as dots at the periphery of the pancreatic buds. these stainings corresponded to small developing lumina, called here secondary lumina. three-dimensional confocal reconstructions were generated to determine the spatial relationship between these secondary lumina and the central duct lumen. no connection between the central and the peripheral lumina were detected, indicating that the latter are blind and independent from the central lumen. furthermore, a section through the e pancreatic bud stained for e-cadherin and mucin- expression showed that the cells of the outer layer are morphologically different from internal cells. the outer cells had a cylindrical shape and a basal nucleus. their apical pole faced the peripheral lumina, as shown by mucin- staining. in addition, the localization of two intracellular markers in this outer epithelial layer was typical of polarized cells. the golgi marker gm- was found on the apical side of the nucleus, and the microtubule-organizing center detected by pericentrin staining was located in the subapical region of the cells. in contrast, except for the cells that lined the central duct lumen, the internal cells of the pancreatic bud showed no mucin- expression and the localization of the golgi and mtoc was random. altogether, these data indicated that the pancreatic bud is a compact epithelial mass, in which the cells are not polarized, except for the outer cells which all have basal and lateral membrane domains; some cells also posses an apical domain. to characterize the acquisition of cell polarity during the transition of a globular pancreatic bud to the monolayered epithelium lining the primitive ducts, we analyzed the formation of tight junctions. the latter play a role in cell adhesion and in the establishment and maintenance of apico-basal polarity. to this end, we studied the localization of the tight junction-specific protein zo- from e to e. despite the continuous presence of adherens junctions between pancreatic epithelial cells, strong zo- staining was not detected in the pancreas before e. from that stage on, zo- was progressively seen in cells facing a lumen. therefore, zo- localization at the tight junctions was subsequent to the onset of polarization of peripheral cells in the e pancreatic bud and was concomitant with the remodeling of the epithelial bud into a monolayered epithelium lining the primitive duct lumina. a similar epithelial transition, from a mass to monolayers, also occurs during submandibular gland morphogenesis. as shown in figure 3b, zo- staining was not visible in the early stage of smg morphogenesis and only became visible after remodeling of the epithelial mass into monolayers lining a lumen. high magnification pictures of epithelial cell monolayers clearly demonstrated that zo- separates the apical domain from the lateral domain. sdf- and its receptor cxcr are expressed in developing pancreas and smg the mesenchyme is a source of morphogenesis-inducing signals in the pancreas and in the smg. therefore, we looked for mesenchymal factors that could regulate epithelial morphogenesis in these organs and considered sdf- as a candidate. indeed, this chemokine and its g protein-coupled receptor cxcr have been proposed to control cell movement in many developing organs, including the endoderm during zebrafish gastrulation. at the late stages of pancreas development, this signaling pathway is involved in pancreatic cell migration and β-cell survival. we first investigated the expression pattern of sdf- and cxcr in the pancreas by performing in situ hybridization experiments. we focused on embryos at e, when epithelial cells are organized in a globular mass, and e, when remodeling has been initiated. we found that sdf is mainly expressed in the mesenchyme, with lower staining intensity in the epithelium; cxcr was found in epithelial cells, as early as e. sdf and cxcr expression was also found in the vasculature. this expression pattern was maintained up to e, a stage at which sdf expression in the central most region of the epithelium became intense. in the developing smg, the expression of sdf and cxcr was comparable to that in the pancreas. from e to e: sdf was expressed in the mesenchyme and cxcr in the epithelium. moreover, cxcr the second receptor for sdf- was observed by immunolocalization in endothelial structures of the smg, but not of the pancreas. from this set of expression data, we concluded that the spatial and temporal expression pattern of sdf- and cxcr in the pancreas and smg suggests a potential role for sdf- signaling in their development. sdf- signaling controls pancreatic branching morphogenesis to address the role of sdf- signaling in epithelial morphogenesis, we resorted to explants of e pancreas cultured on filters. pancreatic cultures have only been validated for analyzing pancreatic cell differentiation when investigating the role of extracellular factors. therefore, we first validated the pancreatic explants as a model for studying branching epithelial morphogenesis. at the start of the culture the explants presented as an epithelial cell mass surrounded by mesenchyme, showing early signs of branching and small, independent, lumina. after to days of culture, the epithelial mass was remodeled into tubulo-acinar epithelial monolayers imbricated within the mesenchyme, as described in vivo. most epithelial cells had acquired polarity with the apical pole facing a well-defined lumen, as revealed by mucin- staining, and the basal pole facing the mesenchyme. we concluded that cultured pancreatic explants provide a model to analyze the formation of the primitive ductal network. to investigate if sdf- controls epithelial morphogenesis in developing pancreas, we performed gain- and loss-of-function experiments by treating cultured explants with exogenous recombinant sdf- and with a specific pharmacological inhibitor of cxcr. pancreatic explants cultured for days were analyzed by immunofluorescence with antibodies against e-cadherin and mucin- no effect of exogenous sdf- on epithelial organization was observed as compared to control explants. the absence of effect of exogenous sdf- is likely to result from the presence of endogenous sdf- whose activity cannot be amplified. in contrast, we found that upon amd treatment, morphogenesis was impaired. epithelial monolayers were not formed and the epithelial cells remained clustered. the mesenchyme was not imbricated with the epithelium and remained at the periphery of the explants. the lumina, as revealed by mucin- staining, were smaller and often surrounded by multiple layers of epithelial cells. identical results were obtained with explants from e embryos, and the effect of amd was dose-dependent, since milder anomalies were observed at μm than at μm. thus, at the end of the culture, the amd3100-treated explants resembled the e pancreas before the onset of remodeling into a monolayered epithelium. amd treatment did not change the proliferation or the apoptosis index and this was supported by the normal size of these explants. immunostaining with antibodies against insulin, glucagon or carboxypeptidase-a revealed normal differentiation in the explants treated with amd. from these experiments we concluded that sdf-1/cxcr signaling is potentially required for the remodeling of the epithelial cell mass into primitive pancreatic ducts lined by a monolayered epithelium. sdf- signaling controls smg branching morphogenesis to investigate if sdf- modulates branching morphogenesis of other exocrine glands, we addressed the role of sdf- signaling in smg epithelial morphogenesis. as shown above, this organ develops according to the same two-stage epithelial transition as in the pancreas. we resorted to cultures of e embryonic explants, as described earlier by others. the smg epithelial bud surrounded by its mesenchyme was excised and cultured on filter for up to days in the presence of sdf- and sdf- antagonists. similarly to the pancreas, gain-of-function experiments with exogenous sdf- did not affect smg branching. however, upon amd treatment, morphogenesis of the smg was impaired. the size of the glands was smaller and the number of epithelial buds was reduced, as compared to control glands, indicating that sdf- signaling is required for branching. quantification of the number of buds in amd3100-treated explants revealed a reduction in the budding index, as early as one day after the onset of the treatment, and culminating at 80% reduction after days, as compared to control cultures. as cxcr was also expressed in the blood vessels surrounding the smg, the sdf- antagonist ccx which prevents sdf- from binding to cxcr was also tested. as shown in figure 6a, ccx inhibited smg morphogenesis by reducing the number of epithelial buds. this was confirmed by a 60% reduction in the budding index as compared to controls after days. these experiments suggested a role for sdf- signaling via cxcr and cxcr in the branching morphogenesis of the smg. we further investigated the morphogenic defect observed by the addition of amd to the culture. as sdf- signaling can control both cell survival and cell proliferation in different cell types, blocking sdf- binding to cxcr with amd could explain the reduced size of the glands. therefore, we performed immunodetection of phosphohistone h a marker of mitosis, and of activated caspase a marker of cell apoptosis, in control, amd3100- and ccx733-treated explants. quantification of these stainings revealed that proliferation was not affected when sdf- signaling was abrogated. however, apoptosis showed a three-fold increase in the epithelial buds of amd3100-treated explants. this observation could explain the reduced size of the smg epithelium. we then assessed whether the increase in apoptosis was the cause of the branching defect. for this, we cultured explants in the presence of amd alone or in combination with a general inhibitor of caspases. apoptosis was effectively prevented as revealed by the near complete absence of activated caspase staining on sections from caspase inhibitor-treated explants. however, in the explants co-cultured with amd and the caspases inhibitor, the branching defect was still observed, indicating that the reduced branching activity is not a consequence of increased apoptosis. since sdf- did not affect cell survival in developing pancreas but did control branching morphogenesis of the pancreas, we conclude that sdf- has differential effects on organ development, but that it has common effects on epithelial branching. genetic deficiency of cxcr or sdf- transiently affects epithelial morphogenesis to verify in vivo the relevance of our ex vivo observations, we analyzed the morphogenesis of embryonic pancreas in sdf knockout mice. at e the ventral pancreas in sdf knockouts showed less branching and had a more globular shape as compared to controls, confirming the role of sdf- in branching morphogenesis. the degree of branching was evaluated by counting the number of buds and clefts in control and knockout ventral pancreas. this quantification revealed a two-fold decrease in the number of buds in the absence of sdf-. however, the dorsal pancreas did not show obvious branching defects, but was more elongated than in controls and it adopted a hammerhead-like structure while the controls were more rounded. the branching defect was transient since e -e sdf1-/- embryos did not show deficient morphogenesis. since the consequences of sdf ablation in vivo were rather modest as compared to the effects of sdf inhibition in vitro, we looked at cxcr4-/- pancreas. at e and e, the dorsal and ventral pancreata were not significantly different in cxcr4-/- embryos and in wild-type animals. however, when cultured on filters cxcr4-/- pancreatic explants showed a strong morphogenic deficiency, similar to that seen when wild-type explants were cultured in the presence of amd a sdf- antagonist. this similarity proves that amd specifically targets and inhibits sdf- signaling via the cxcr receptor. taken together, these data indicated that sdf- controls pancreas morphogenesis in vivo, but also suggest that compensatory effects operating in vivo mask part of the role of sdf- we also analyzed the development of smg in cxcr4-/- and sdf1-/- embryos, but the smg phenotypes appeared normal, both in vitro and in vivo. we concluded that no genetic evidence for a role of sdf- signaling in smg branching could be collected. however, the existence of compensatory mechanisms for genetic deficiencies in the pancreas suggests that compensation may also occur in the smg. discussion in this paper we show that the primitive pancreatic ductal network, which is lined by a monolayered and polarized epithelium, forms by remodeling of a globular mass of non polarized epithelial cells. sdf- is expressed in the mesenchyme surrounding the epithelium and its receptor cxcr is found in the epithelial cells of the pancreas. sdf- signaling promotes branching morphogenesis of the pancreas in vitro and in vivo. we also provide in vitro evidence that smg morphogenesis is controlled by sdf- previous models depicting pancreatic duct morphogenesis in mammals suggest that it results from the branching of a polarized epithelium. we show here that formation of the primitive ductal network is not initiated by iterative branching of a tubular structure extending from the gut. the pancreatic bud at e -e consists of a globular mass of epithelial cells with a central duct lumen. this lumen, connected to the gut tube lumen, forms extensions within the pancreatic bud, and is surrounded by several layers of cells. these cells are linked by adherens junctions, but lack tight junctions. at this stage, we observe that some cells at the periphery of the mass show apico-basal polarity and delineate blind lumina. based on these observations, we propose that primitive duct branches arise from extensions of the central gut-derived duct that fuse with secondary lumina that appear at e. interestingly, this process bears resemblance with lumen development in the zebrafish gut tube and exocrine pancreas. indeed, in these organs the lumen arises by fusion of multiple lumina. after the formation of primitive ducts, further branching and expansion of the pancreatic ducts occur by iterative lateral branching, as illustrated by puri and hebrok. this is also similar to the development of the mouse smg, where the epithelial cells first form a mass that becomes remodeled into a monolayered epithelium. like in the pancreas, epithelial cells in the smg express e-cadherin and β-catenin. zo- is only detectable when cells are lining a lumen. the time-course and 3-d analysis of embryonic pancreas development indicated that an initial phase of polarization occurs in cells of the outermost layer of the pancreatic bud at e. this precedes remodeling of the bud, suggesting that, at the onset of pancreas development, remodeling may be triggered by the acquisition of polarity and not the reverse. beyond this stage, acquisition of apico-basal polarity and primitive duct formation cannot be dissociated morphologically but must be orchestrated by several regulators acting on distinct aspects. indeed, in our culture explants, blocking sdf- signaling did not prevent epithelial cells in the mass to acquire apical characteristics such as assembly of tight junction complexes and primary cilia. nevertheless, this polarization program could not go to completion, as the cells remained clustered and most of them did not acquire a basal domain. during the remodeling of the pancreatic globular mass, the epithelium is in contact with the mesenchyme. the latter tissue has been proposed to provide signals that control epithelial proliferation, differentiation and morphogenesis. we have identified here sdf-1/cxcr signaling as a potential regulator of branching morphogenesis. indeed, sdf- and its receptor, cxcr are expressed in embryonic pancreas in a complementary fashion. when epithelial cells are still organized as a non polarized mass, sdf- is expressed in the mesenchyme whereas cxcr is localized in the epithelium. moreover, when sdf- signaling is inhibited by pharmacological means or by cxcr genetic deficiency, pancreas morphogenesis is perturbed. these data are consistent with a model in which the mesenchymally-produced sdf- promotes epithelial remodeling and thus primitive duct morphogenesis. while this work was under review, ueland and collaborators demonstrated that sdf- controls epithelial kidney morphogenesis in vitro. our data in the pancreas and smg, together with those of ueland et al. demonstrate the role of sdf- in epithelial branching morphogenesis. the molecular nature of the branching defects does not involve a change in the proliferation or apoptosis indexes, but may be explained by a change in the migration potential of epithelial cells. there is no perfect overlap between the results obtained with the pharmacological inhibition and with genetic inactivation of sdf- signaling in the pancreas. pharmacological inhibition of sdf- signaling in vitro severely repressed branching morphogenesis, but genetic deficiency in sdf1-/- pancreas showed more modest effects in vivo. also, the effect of cxcr gene ablation only led to abnormal pancreas morphogenesis when the organ was cultured in vitro. we suggest that compensatory pathways may be active in vivo but not in vitro. in our culture system, endothelial cells are present and organized around the epithelium like in vivo, the main difference being that there is no blood circulation in the endothelial network in vitro. we suggest that signal coming directly or indirectly from the circulation could participate in the control of epithelial branching morphogenesis of the pancreas and smg. such a model would further extend the role of the vasculature in pancreas development, since others previously showed that blood flow through the aorta is required for dorsal pancreas budding from the endoderm. in developing smg, cxcr is expressed in the epithelium and sdf- in the mesenchyme, like in the pancreas. this suggests that sdf- controls branching morphogenesis by a direct mechanism on the epithelium. importantly, cxcr is detected in endothelial cells of the smg, but not in the pancreas. in smg explants, we observed a branching defect when sdf- binding to cxcr was inhibited by ccx this suggests that sdf- can also indirectly control epithelial branching morphogenesis, via the endothelium. activation of cxcr in the endothelium may induce the production of an unidentified factor which in turn would signal to the epithelium and act in parallel to or amplify the direct sdf- effects on epithelial morphogenesis. in the smg we also noticed a difference between the effects of pharmacological inhibition of sdf- and genetic deficiencies induced by inactivation of the sdf or cxcr genes. in vitro and in vivo development of cultured cxcr4-/- and sdf1-/- smg was normal, unlike development of amd3100- or ccx733-treated explants. this suggests the existence of other ligands that could bind cxcr or alternative signaling pathways with which sdf-1/cxcr4/ might interact in this organ. it has been reported that migrating muscle progenitors cells reach the anlage of the tongue in cxcr4-/- or gab1-/-, but not in the double-mutant mice. a crosstalk between g-protein-coupled receptors and tyrosine kinase receptors may contribute to compensatory mechanisms. finally, when sdf- signaling was blocked, the size of the epithelium was reduced in the smg but not in the pancreas. although sdf- is important for survival of insulin-producing β-cells, we did not observe any effect of sdf- signaling on cell survival early in pancreas development, even when e explants were cultured in the absence of serum. this indicates that the survival signals are different in the two organs. a role for sdf- in cell survival has already been described in several systems. we excluded the possibility that the role of sdf- in cell survival in smg coincided with its role in branching morphogenesis, thereby indicating that sdf- plays distinct roles in the smg.  in conclusion, the present work shows that the formation of primitive ducts in the pancreas depends on remodeling of a globular mass of epithelial cells, as described earlier in smg. our data also identify sdf- as a factor that may promote the transition from a mass of epithelial cells towards monolayers in the pancreas and smg. understanding the mechanisms of such epithelial transition is relevant to carcinogenesis, which is associated with loss of epithelial polarity and formation of a mass of cells that fills the ductal lumen. 
the morphogenic mechanisms by which the ductal network is established as well as the signaling pathways involved in this process are poorly understood. in conclusion, our data show that the primitive pancreatic ductal network, which is lined by a monolayered and polarized epithelium, forms by remodeling of a globular mass of non polarized epithelial cells. during the first transition, the monolayered and polarized endodermal cells give rise to tissue buds composed of a mass of non polarized epithelial cells. reorganization of a globular mass of epithelial cells in polarized monolayers is also observed during submandibular glands development. reorganization of cultured pancreatic buds into monolayered epithelia was blocked in the presence of amd a sdf- antagonist. by morphological analyzis of wild-type and mutant mouse embryos and using cultured embryonic explants we investigated how epithelial morphogenesis takes place and is regulated by chemokine signaling. the exocrine pancreas is composed of a branched network of ducts connected to acini. pancreas ontogenesis displayed a sequence of two opposite epithelial transitions. analyzis of sdf and cxcr knockout embryos at the stage of the second epithelial transition revealed transient defective morphogenesis of the ventral and dorsal pancreas. our data also suggest that sdf- controls the branching morphogenesis of several exocrine tissues. during the second transition the buds reorganize into branched and polarized epithelial monolayers that further differentiate into tubulo-acinar glands.
 the exocrine pancreas is composed of a branched network of ducts connected to acini. they are lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesoderm-derived mesenchyme. the morphogenic mechanisms by which the ductal network is established as well as the signaling pathways involved in this process are poorly understood.  by morphological analyzis of wild-type and mutant mouse embryos and using cultured embryonic explants we investigated how epithelial morphogenesis takes place and is regulated by chemokine signaling. pancreas ontogenesis displayed a sequence of two opposite epithelial transitions. during the first transition, the monolayered and polarized endodermal cells give rise to tissue buds composed of a mass of non polarized epithelial cells. during the second transition the buds reorganize into branched and polarized epithelial monolayers that further differentiate into tubulo-acinar glands. we found that the second epithelial transition is controlled by the chemokine stromal cell-derived factor - the latter is expressed by the mesenchyme, whereas its receptor cxcr is expressed by the epithelium. reorganization of cultured pancreatic buds into monolayered epithelia was blocked in the presence of amd a sdf- antagonist. analyzis of sdf and cxcr knockout embryos at the stage of the second epithelial transition revealed transient defective morphogenesis of the ventral and dorsal pancreas. reorganization of a globular mass of epithelial cells in polarized monolayers is also observed during submandibular glands development. we found that sdf- and cxcr are expressed in this organ and that amd treatment of submandibular gland explants blocks its branching morphogenesis.  in conclusion, our data show that the primitive pancreatic ductal network, which is lined by a monolayered and polarized epithelium, forms by remodeling of a globular mass of non polarized epithelial cells. our data also suggest that sdf- controls the branching morphogenesis of several exocrine tissues. 