In vitro validation of duct differentiation in developing embryonic mouse pancreas.
BACKGROUND
Early embryonic pancreatic epithelia have the capacity for either endocrine or exocrine lineage commitment. Recent studies demonstrated the pluripotential nature of these undifferentiated cells. Isolated pancreatic epithelia grown under the renal capsule formed primarily islets. However, when these same epithelia were grown in a basement-membrane-rich gel (Matrigel) they formed mostly ducts. Currently, there is no model for in vitro pancreatic duct formation and therefore, the mechanism of duct morphogenesis has never been described. The purpose of this study was to provide such a model by characterizing the expression of two duct markers, carbonic anhydrase II (CAII) and the cystic fibrosis transmembrane conductance regulator (CFTR), in isolated undifferentiated pancreatic epithelia grown in vitro.


METHODS
We microdissected embryonic pancreases at Embryonic Days (E)9.5-11.5 and performed RT-PCR for CAII and CFTR on E9.5 whole pancreases, E10. 5 and E11.5 epithelia, as well as E11.5 epithelia grown for 7 days in Matrigel. Next we performed in situ hybridization for CAII and CFTR and immunohistochemistry for CAII on E11.5 epithelia grown for 7 days in Matrigel.


RESULTS
Early, undifferentiated embryonic pancreatic epithelium does not express CAII and CFTR by RT-PCR. When E11.5 epithelia were grown for 7 days in Matrigel, however, gene expression for both markers is upregulated as ducts form. Furthermore, CAII was seen by IHC and both CAII and CFTR were seen by in situ hybridization in the ducts after 7 days in Matrigel.


CONCLUSIONS
These data validate our in vitro system as a model for studying the mechanism of normal pancreatic duct differentiation and may potentially help us to understand the faulty mechanism involved in pancreatic ductal carcinogenesis.
Kadison
A S
AS
New York University Medical Center, New York, New York, USA.
Maldonado
T S
TS
Crisera
C A
CA
Longaker
M T
MT
Gittes
G K
GK
D000818:Animals; D015415:Biomarkers; D002256:Carbonic Anhydrases / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods* / Q000592:standards*; D002454:Cell Differentiation / Q000502:physiology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D005260:Female; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D017403:In Situ Hybridization; D051379:Mice; D008815:Mice, Inbred Strains; D010183:Pancreatic Ducts / Q000166:cytology* / Q000196:embryology*; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015415:Biomarkers; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D002256:Carbonic Anhydrases