Ucn 3 is expressed in alpha and beta cells in freshly fixed human islets.

<p>Serial sections of the same human islet demonstrate that Ucn 3 expression co-localizes with insulin (A) and glucagon (B), but not somatostatin. The same islet shows no immunoreactivity for the related peptide Ucn 1 using an antiserum similar to the one used for the detection of Ucn 3 (C, D). Additional controls stained with secondary antibody only are negative as well (E, F). High power magnifications illustrate that the large majority of beta (G) and alpha (H) cells co-express Ucn 3 (arrows), although occasional insulin⁺ and glucagon⁺ cells can be found that do not stain for Ucn 3 (asterisks in G and H). Ucn 3 gene expression in FACS-purified dissociated human islet cells (I) from three individual donors confirmed that Ucn 3 is enriched in human beta (J) and alpha cells (K). The acinar cell fraction was depleted for the expression of all endocrine markers (L). Gene expression is normalized to HPRT and expressed relative to pre-sort levels. Error bars indicate standard errors, asterisks reflect significant differences at a p-value <0.05 as determined by t-test.</p

Ucn 3 expression in freshly fixed human islets co-localizes with PC1/3 in beta cells but not in alpha cells.

<p>Ucn 3 expression in human insulin+ beta cells coincides with PC1/3 (A), while Ucn 3+ glucagon+ alpha cells do not express PC1/3 (B). Arrows in all panels indicate Ucn 3⁺ alpha cells, whereas asterisks denote Ucn 3⁺ mature beta cells.</p

Ucn 3 expression is acquired upon implantation of polyhormonal cells that differentiate primarily into alpha cells.

<p>Implantation of hESC-derived pancreatic cultures, either non-enriched (A) or enriched for pancreatic endoderm by CD142 (B) in the epidydimal fat pad for 9 weeks supports the formation of alpha and beta cells that co-express Ucn 3. In contrast, implantation of CD318-enriched polyhormonal cells leads to the formation of predominantly alpha cells that co-express Ucn 3, with no detectable expression of insulin (C).</p

Ucn 3 expression in the primate pancreas is a feature of alpha and beta cells.

<p>Ucn 3 immunoreactivity co-localizes with insulin as well as glucagon in both human (A, B) and macaque (C, D) pancreas.</p

Ucn 3 expression in hESC-derived beta cells co-localizes with PC1/3.

<p>Ucn 3 in beta cells differentiated in implants of hESC-derived pancreatic endoderm cells co-labels with PC1/3 (A, B), while Ucn 3 in glucagon⁺ alpha cells does not (C, D). Arrows in all panels indicate Ucn 3⁺ alpha cells, whereas asterisks denote Ucn 3⁺ mature beta cells. Pound signs indicate insulin⁺, PC1/3⁺ Ucn 3^- immature beta cells. Boxed areas in the first row of images are magnified in subsequent panels.</p

Expression of Ucn 3 in hESC-cell derived alpha and beta cells requires <i>in vivo</i> maturation.

<p>Quantitative PCR analysis of key differentiation stages towards the human beta cell lineage reveals no Ucn 3 expression at the embryonic stem cell (ES) or definitive endoderm (DE) stage. While pancreatic cultures enriched for pancreatic endoderm cells (PE) by CD142 did not express Ucn 3, polyhormonal cells (PH) enriched from the same cultures by CD200, started to express Ucn 3. Implants of hESC-derived pancreatic progenitors robustly express Ucn 3 after differentiation and maturation <i>in vivo</i> at 140 days post-transplant (Tx). Ucn 3 expression is measured by qRT-PCR, normalized to TATA-box binding protein (TBP) and plotted relative to ES cells (A). These observations are confirmed on <i>in vitro</i> differentiated hESC-derived pancreatic endoderm, which contains a minor fraction of polyhormonal cells that express insulin (B) and glucagon (C), but are mostly devoid of Ucn 3 immunoreactivity with the exception of faint Ucn 3 expression in the occasional glucagon⁺ cell (C, inset). Ucn 3 immunoreactivity is robustly upregulated 18 weeks following engraftment in the epidydimal fat pads of mice and demonstrates significant Ucn 3 colocalization with insulin⁺ beta cells and glucagon⁺ alpha cells (D). Ucn 3 immunoreactivity is robustly upregulated 140 days post implantation in TheraCyte encapsulation devices and co-localizes with both insulin (E) and glucagon (F). Heterogeneity exists among both alpha and beta cells regarding the expression of Ucn 3. Arrows indicate Ucn 3⁺ cells that co-express insulin (G) or glucagon (H).</p

Ucn 3 expression trails the appearance of insulin in ontogeny.

<p>Ucn 3 expression, undetectable by immunohistochemistry at E12.5 (A, B) and E15.5 (C, D), first appears in a subset of beta cells around E17.5 of embryonic development (E, F). The fraction of Ucn 3⁺ beta cells increases perinatally (G, H) and by p7, the majority of beta cells (I, J), including those appearing as singlets in the acinar tissue (K) are clearly Ucn 3⁺. From p14 onwards, Ucn 3 expression is evident in all beta cells (L–O).</p

Primer information.

<p>Primer information.</p

Expression and Functional Characterization of Membrane-Integrated Mammalian Corticotropin Releasing Factor Receptors 1 and 2 in <i>Escherichia coli</i>

<div><p>Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2β as well as fragments lacking the N-terminal extracellular domain, were overproduced in <i>E. coli</i>. The characteristics of different CRFR2β -PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1α and CRFR2β expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of <i>E. coli</i> bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2β product was obtained in comparison to CRFR1α. Following a detergent screen analysis, bacterial membranes containing CRFR1α and CRFR2β were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1α and CRFR2β membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our <i>E. coli</i> expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.</p></div

Alkaline phosphatase fusion protein analysis in <i>E. coli</i> of mCRFR2β.

<p>(<b>A</b>) Specifically designed C-terminally truncated versions of mCRFR2β fused to bacterial membrane topology reporter alkaline phosphatase (PhoA) confer different phenotypes at the level of colony color. PhoA activity was assessed qualitatively by visual inspection of the colonies. (<b>B</b>) The bacterial colony colors conferred by these different protein fusions are in agreement with the hepta-helical transmembrane model of mCRFR2β. The aa numeration refers to the native receptors pre-protein sequence.</p