ABSTRACT
BACKGROUND/PURPOSE: Many studies in pediatric surgical research use a quantitative analysis of gene expression in microscopic quantities of tissue. The authors describe an analysis of the beta-tubulin mRNA content of the embryonic pancreas, which contains abundant endogenous RNases. A detailed analysis of this RNase-containing system will provide a good template for analysis of other potentially simpler systems. METHODS: Embryonic mouse pancreases were harvested at serial gestational ages. DAPI nuclear staining allowed for counting of cells. cDNA was amplified using a fluoresceinated primer and the normalized fluorescence determined. Known numbers of molecules were amplified in parallel as a standard control. RESULTS: The number of cells increased from 38,000 to 2,700,000 between embryonic day 10.5 (E10.5) and E18.5. mRNA for beta-tubulin did not increase proportionately. Assuming a yield of 100% at E10.5 when no RNases are present, the yield of expected mRNA was 65.3% at E12.5, 13.8% at E15.5, and 0.9% at E18.5, presumably because of the appearance of RNases. CONCLUSIONS: Several parameters must be considered in performing semiquantitative reverse transcription polymerase chain reaction: (1) the yield of RNA based on the projected amount of mRNA, (2) the number of cells in the tissue, and (3) a known number of template molecules amplified in parallel.
Subject(s)
Pancreas/enzymology , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleases/analysis , Tubulin/analysis , Animals , Cell Count , Cell Division , Gene Expression Regulation, Developmental , Gestational Age , Insulin/analysis , Insulin/metabolism , Mice , Pancreas/cytology , Pancreas/embryology , RNA, Messenger/metabolism , Tubulin/metabolismABSTRACT
BACKGROUND/PURPOSE: Retinoid signaling plays an important role in many differentiation pathways. Retinoid signaling has been implicated in the induction of differentiation by pancreatic ductal cancer cell lines and in patients with pancreatic cancer. The authors wished to better understand the role of retinoid signaling in pancreatic development. METHODS: Embryonic pancreas was harvested from mice at serial gestational ages and immunohistochemical analysis was performed for retinoic acid receptors (RAR-alpha, RAR-beta, RAR-gamma), and retinoid X receptors (RXR-alpha, RXR-beta, and RXR-gamma). Also, early embryonic pancreases were cultured for 7 days with exogenous 9-cis retinoic acid (9cRA) or all-trans retinoic acid (atRA) and analyzed histologically and immunohistochemically. RESULTS: Retinoid receptors were expressed in a lineage-specific distribution, with stronger expression for many in the exocrine compartment. The receptors were not often expressed until late gestation. Exogenous 9cRA induced predominantly ducts instead of acini, plus more mature endocrine (islet) architecture. Exogenous atRA induced predominantly acini instead of ducts, with no apparent endocrine effect. CONCLUSIONS: Retinoids may have an important role in pancreatic differentiation, with a particular effect on secondary lineage selection between ductal and acinar phenotype. Because the control of ductal versus acinar differentiation has been implicated strongly in the pathogenesis of pancreatic ductal carcinoma, these results may lay the groundwork for studies in the mechanism of induced differentiation of pancreatic ductal cancer by retinoids.
Subject(s)
Embryonic and Fetal Development/physiology , Pancreas/embryology , Receptors, Retinoic Acid/analysis , Signal Transduction/physiology , Tretinoin/pharmacology , Animals , Cell Lineage , Female , Immunohistochemistry , Mice , Mice, Inbred Strains , Models, Animal , Organ Culture Techniques , Pregnancy , Pregnancy, Animal , Reference Values , Sensitivity and Specificity , Tretinoin/metabolismABSTRACT
In this study, we establish the presence of a unique member of the PRL-like protein-C (PLP-C) subfamily in the mouse, PLP-C alpha, characterize its complementary DNA and gene, and map its chromosomal location and pattern of expression during pregnancy. Mouse PLP-C alpha encodes for a 239 amino acid protein and possesses from 69-71% identity with rat PLP-C, PLP-Cv, PLP-D, and PLP-H. Another feature characteristic of PLP-C subfamily members that is also present in mouse PLP-C alpha is a 6-exon/5-intron gene structure including an aromatic domain encoded by exon 3. Southern analysis with mouse and rat PLP-C subfamily probes suggested the existence of a single mouse PLP-C alpha gene. Mouse PLP-C alpha maps to chromosome 13 along with other members of the mouse PRL family. Expression of mouse PLP-C alpha increases dramatically as gestation advances and is restricted to spongiotrophoblast and trophoblast giant cells of the junctional zone. In summary, we have established the presence of a new PLP-C subfamily member in the mouse and demonstrated its similarity in structure and expression to rat PLP-C subfamily members. This level of conservation between species expands the biological significance of the PLP-C subfamily and provides additional opportunities for genetically evaluating its function.
