Jonas Ahnfelt-Rønne

An Improved Method for Three-dimensional Reconstruction of Protein Expression Patterns in Intact Mouse and Chicken Embryos and Organs

We have developed a wholemount immunofluorescence protocol for the simultaneous detection of up to three proteins in mouse and chicken embryos. Combined with Murrays clearing reagent (BABB) and microscope objectives with long working ranges and high numerical apertures mounted on a confocal microscope, cellular resolution can be obtained in depths offering the possibility of examining expression patterns in entire organs or embryos. Three-dimensional projections of the optical confocal sections can be computed with computer software allowing rotation around any axis. The protocol is robust and we find that most antibodies working on tissue sections also work with this protocol. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Mind bomb 1 is required for pancreatic β-cell formation

During early pancreatic development, Notch signaling represses differentiation of endocrine cells and promotes proliferation of Nkx6-1+Ptf1a+ multipotent progenitor cells (MPCs). Later, antagonistic interactions between Nkx6 transcription factors and Ptf1a function to segregate MPCs into distal Nkx6-1−Ptf1a+ acinar progenitors and proximal Nkx6-1+Ptf1a− duct and β-cell progenitors. Distal cells are initially multipotent, but evolve into unipotent, acinar cell progenitors. Conversely, proximal cells are bipotent and give rise to duct cells and late-born endocrine cells, including the insulin producing β-cells. However, signals that regulate proximodistal (P-D) patterning and thus formation of β-cell progenitors are unknown. Here we show that Mind bomb 1 (Mib1) is required for correct P-D patterning of the developing pancreas and β-cell formation. We found that endoderm-specific inactivation of Mib1 caused a loss of Nkx6-1+Ptf1a− and Hnf1β+ cells and a corresponding loss of Neurog3+ endocrine progenitors and β-cells. An accompanying increase in Nkx6-1−Ptf1a+ and amylase+ cells, occupying the proximal domain, suggests that proximal cells adopt a distal fate in the absence of Mib1 activity. Impeding Notch-mediated transcriptional activation by conditional expression of dominant negative Mastermind-like 1 (Maml1) resulted in a similarly distorted P-D patterning and suppressed β-cell formation, as did conditional inactivation of the Notch target gene Hes1. Our results reveal iterative use of Notch in pancreatic development to ensure correct P-D patterning and adequate β-cell formation.

Ptf1a-mediated control of Dll1 reveals an alternative to the lateral inhibition mechanism

Neurog3-induced Dll1 expression in pancreatic endocrine progenitors ostensibly activates Hes1 expression via Notch and thereby represses Neurog3 and endocrine differentiation in neighboring cells by lateral inhibition. Here we show in mouse that Dll1 and Hes1 expression deviate during regionalization of early endoderm, and later during early pancreas morphogenesis. At that time, Ptf1a activates Dll1 in multipotent pancreatic progenitor cells (MPCs), and Hes1 expression becomes Dll1 dependent over a brief time window. Moreover, Dll1, Hes1 and Dll1/Hes1 mutant phenotypes diverge during organ regionalization, become congruent at early bud stages, and then diverge again at late bud stages. Persistent pancreatic hypoplasia in Dll1 mutants after eliminating Neurog3 expression and endocrine development, together with reduced proliferation of MPCs in both Dll1 and Hes1 mutants, reveals that the hypoplasia is caused by a growth defect rather than by progenitor depletion. Unexpectedly, we find that Hes1 is required to sustain Ptf1a expression, and in turn Dll1 expression in early MPCs. Our results show that Ptf1a-induced Dll1 expression stimulates MPC proliferation and pancreatic growth by maintaining Hes1 expression and Ptf1a protein levels.

Preservation of proliferating pancreatic progenitor cells by Delta-Notch signaling in the embryonic chicken pancreas

BackgroundGenetic studies have shown that formation of pancreatic endocrine cells in mice is dependent on the cell autonomous action of the bHLH transcription factor Neurogenin3 and that the extent and timing of endocrine differentiation is controlled by Notch signaling. To further understand the mechanism by which Notch exerts this function, we have investigated pancreatic endocrine development in chicken embryos.ResultsIn situ hybridization showed that expression of Notch signaling components and pro-endocrine bHLH factors is conserved to a large degree between chicken and mouse. Cell autonomous inhibition of Notch signal reception results in significantly increased endocrine differentiation demonstrating that these early progenitors are prevented from differentiating by ongoing Notch signaling. Conversely, activated Notch1 induces Hes5-1 expression and prevents endocrine development. Notably, activated Notch also prevents Ngn3-mediated induction of a number of downstream targets including NeuroD, Hes6-1, and MyT1 suggesting that Notch may act to inhibit both Ngn3 gene expression and protein function. Activated Notch1 could also block endocrine development and gene expression induced by NeuroD. Nevertheless, Ngn3- and NeuroD-induced delamination of endodermal cells was insensitive to activated Notch under these conditions. Finally, we show that Myt1 can partially overcome the repressive effect of activated Notch on endocrine gene expression.ConclusionWe conclude that pancreatic endocrine development in the chicken relies on a conserved bHLH cascade under inhibitory control of Notch signaling. This lays the ground for further studies that take advantage of the ease at which chicken embryos can be manipulated.Our results also demonstrate that Notch can repress Ngn3 and NeuroD protein function and stimulate progenitor proliferation. To determine whether Notch in fact does act in Ngn3-expressing cells in vivo will require further studies relying on conditional mutagenesis.Lastly, our results demonstrate that expression of differentiation markers can be uncoupled from the process of delamination of differentiating cells from the epithelium.

Mesenchymal Bone Morphogenetic Protein Signaling Is Required for Normal Pancreas Development

OBJECTIVE Pancreas organogenesis is orchestrated by interactions between the epithelium and the mesenchyme, but these interactions are not completely understood. Here we investigated a role for bone morphogenetic protein (BMP) signaling within the pancreas mesenchyme and found it to be required for the normal development of the mesenchyme as well as for the pancreatic epithelium. RESEARCH DESIGN AND METHODS We analyzed active BMP signaling by immunostaining for phospho-Smad1,5,8 and tested whether pancreas development was affected by BMP inhibition after expression of Noggin and dominant negative BMP receptors in chicken and mouse pancreas. RESULTS Endogenous BMP signaling is confined to the mesenchyme in the early pancreas and inhibition of BMP signaling results in severe pancreatic hypoplasia with reduced epithelial branching. Notably, we also observed an excessive endocrine differentiation when mesenchymal BMP signaling is blocked, presumably secondary to defective mesenchyme to epithelium signaling. CONCLUSIONS We conclude that BMP signaling plays a previously unsuspected role in the mesenchyme, required for normal development of the mesenchyme as well as for the epithelium.

Betatrophin: The long awaited circulating factor from the liver promoting β-cell replication?

Regenerative therapy in diabetes with the capacity to reconstitute a functional β-cell mass sufficient for glycemic control holds the promise to effectively prevent the development of devastating late complications due to the unique ability of the β-cell to sense and regulate blood-glucose levels. An ability that cannot be mimicked by insulin replacement therapy or any other means of current treatment regiments for very large patient populations. Recently, Douglas A. Melton’s group from Harvard University reported the identification of a circulating protein secreted from the liver under insulin resistant states which is sufficient to dramatically and specifically increase the replication rate of β-cells in the mouse resulting in an increased functional β-cell mass over time. They re-named the factor betatrophin and described a number of exciting features of this molecule which suggested that it could be a potential candidate for development as a regenerative medicine in diabetes.1 The official name of the gene encoding mouse betatrophin is Gm6484, but it has been annotated a number of times under different names: EG6242192,3, RIFL4, Lipasin5 and ANGPTL8.6 The official human gene name is C19orf80, but it has also been annotated as TD267, LOC559088, as well as RIFL, Lipasin, ANGPTL8 and betatrophin.

A new view of the beta cell

Keywords Alphacell .Betacell .Fluorescentprobe .Functional-mass .Homing .Imaging .Internalisation .IsletofLangerhansAbbreviationsDTBZ DihydrotetrabenazineGLP-1 Glucagon-like peptide 1PET Positron emission tomographyTMEM27 Transmembrane protein 27There is a current unmet need in diabetology for a directmeasure of the functional beta cell mass in patients, partly asa result of a lack of appropriate probes. In this issue ofDiabetologia, Vats and colleagues describe the generationof a new antibody raised against transmembrane protein 27(TMEM27) with promising features for use in beta cellimaging in humans [1].Diabetes mellitus is characterised by high blood glucoselevelscausedbyaninsufficientfunctionalbetacellmass,whichis determined by the actual beta cell mass and how muchinsulin the beta cells produce relative to the physiologicaldemand. Diabetes is a group of metabolic diseases withcompletely different aetiologies; there are three maintypes: type 1 diabetes is caused by autoimmune destructionof the beta cells, resulting in an absolute beta cell deficiency,whereastype2diabetesandgestationaldiabetesarecausedbyarelativebetacelldeficiencyresultingfromperipheralinsulinresistanceandinadequatebetacellfunction,whichisfollowedby loss of beta cell mass in type 2 diabetes.The beta cell mass is plastic, allowing adaptation tophysiological demand. For example, the pancreas is able toincrease the beta cell mass during pregnancy and in obesity,at least in rodents, thus compensating for the increaseddemand for insulin caused by insulin resistance. Diabetesdevelops when this mechanism fails, and it is thereforethought that drugs that improve the capacity to expand thebeta cell mass or, alternatively, prevent the loss of beta cellmass will be of great clinical importance in the treatment ofdiabetes. Evidence from mouse studies has suggested that,even after near complete loss of beta cells, the pancreas hasthe capacity to spontaneously regenerate a sufficiently largefunctional beta cell mass to become independent of insulintreatment [2]. Furthermore, measurable levels of circulatingC-peptide,as well as pancreatic insulin-positive cells,canbefound in type 1 diabetic patients, even many years after theonset of the disease [3]. This is indicative of sustainedformation of new beta cells, lending hope to the notion thatinduction of immune tolerance could offer a potential curefor type 1 diabetes, perhaps in combination with regenerativemedicine.Although beta cell mass declines in both type 1 and type 2diabetes in humans, the process is poorly understood becauseonlyindirectmeansareavailableforestimationofthebetacellmass in vivo. These are based on correlations between func-tional insulin secretion measurements and beta cell mass [4].Furthermore, establishment of the beta cell mass by mor-phometry on biopsies may be grossly misleading owing to

Elucidating the biological roles of insulin and its receptor in murine intestinal growth and function

The role of the intestinal insulin receptor (IR) is not well understood. We therefore explored the effect of insulin (300 nmol/kg per day for 12 days) on the intestine in sex-matched C57Bl/6J mice. The intestinal and metabolic profiles were also characterized in male and female intestinal-epithelial IR knockout (IE-irKO) mice compared with all genetic controls on a chow diet or Western diet (WD) for 4 to 12 weeks. Insulin treatment did not affect intestinal size, intestinal resistance, or metabolic genes, but it reduced proximal-colon crypt depth and acutely increased colonic serine/threonine-specific protein kinase B (AKT) activation. Feeding with a WD increased body weight and fasting insulin level and decreased oral glucose tolerance in C57Bl/6J and IE-irKO mice. However, although the overall responses of the IE-irKO mice were not different from those of Villin-Cre (Vil-Cre):IRfl/+ and IRfl/fl controls, profound differences were found for female control Vil-Cre mice, which demonstrated reduced food intake, body weight, jejunal glucose transport, oral glucose tolerance, and fasting insulin and cholesterol levels. Vil-Cre mice also had smaller intestines compared with those of IE-irKO and IRfl/fl mice and greater insulin-mediated activation of jejunal IR and AKT. In summary, gain- and loss-of-function studies, with and without caloric overload, indicate that insulin did not exert remarkable effects on intestinal metabolic or morphologic phenotype except for a small effect on the colon. However, the transgenic control Vil-Cre mice displayed a distinct phenotype compared with other control and knockout animals, emphasizing the importance of thoroughly characterizing genetically modified mouse models.

Beta cell specific probing with fluorescent exendin-4 is progressively reduced in type 2 diabetic mouse models

Probes based on GLP-1R agonist exendin-4 have shown promise as in vivo β cell tracers. However, questions remain regarding the β cell specificity of exendin-4 probes, and it is unclear if the expression levels of the GLP-1R are affected in a type 2 diabetic state. Using in vivo probing followed by ex vivo imaging we found fluorescent exendin-4 probes to distinctly label the pancreatic islets in mice in a Glp-1r dependent manner. Furthermore, a co-localization study revealed a near 100 percent β cell specificity with less than one percent probing in other analyzed cell types. We then tested if probing was affected in models of type 2 diabetes using the Leprdb/db (db/db) and the Diet-Induced Obese (DIO) mouse. Although nearly all β cells continued to be probed, we observed a progressive decline in probing intensity in both models with the most dramatic reduction seen in db/db mice. This was paralleled by a progressive decrease in Glp-1r protein expression levels. These data confirm β cell specificity for exendin-4 based probes in mice. Furthermore, they also suggest that GLP-1R targeting probes may provide a tool to monitor β cell function rather than mass in type 2 diabetic mouse models.

Quantitative proteomics of intestinal mucosa from male mice lacking intestinal epithelial insulin receptors

The goal of the present study was to determine whether loss of the insulin receptor alters the molecular landscape of the intestinal mucosa, using intestinal-epithelial insulin receptor knockout (IE-irKO) mice and both genetic (IRfl/fl and Villin-cre) controls. Quantitative proteomic analysis by liquid chromatography mass spectrometry was applied to jejunal and colonic mucosa from mice fed a normal chow diet and mice fed a Western diet (WD). Jejunal mucosa from IE-irKO mice demonstrated alterations in all intestinal cell lineages: Paneth, goblet, absorptive, and enteroendocrine cells. Only goblet and absorptive cells were affected in the colon. Also, a marked effect of WD consumption was found on the gut proteome. A substantial reduction was detected in Paneth cell proteins with antimicrobial activity, including lysozyme C-1, angiogenin-4, cryptdin-related sequence 1C-3 and -2, α-defensin 17, and intelectin-1a. The key protein expressed by goblet cells, mucin-2, was also reduced in the IE-irKO mice. Proteins involved in lipid metabolism, including aldose reductase-related protein 1, 15-hydroxyprostaglandin dehydrogenase, apolipoprotein A-II, and pyruvate dehydrogenase kinase isozyme 4, were increased in the mucosa of WD-fed IE-irKO mice compared with controls. In contrast, expression of the nutrient-responsive gut hormones, glucose-dependent insulinotropic polypeptide and neurotensin, was reduced in the jejunal mucosa of IE-irKO mice, and the expression of proteins of the P-type adenosine triphosphatases and the solute carrier-transporter family was reduced in the colon of WD-fed IE-irKO mice. In conclusion, IE-irKO mice display a distinct molecular phenotype, suggesting a biological role of insulin and its receptor in determining differentiated cell specificity in the intestinal epithelium.