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Signe Horn,
Sune Kobberup,
Mette C Jørgensen,
Mark Kalisz,
Tino Klein,
Ryoichiro Kageyama,
Moritz Gegg,
Heiko Lickert,
Jill Lindner,
Mark A Magnuson,
Young-Yun Kong,
Palle Serup, Jonas Ahnfelt-Rønne,
Jan N Jensen
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ABSTRACT: 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.
Proceedings of the National Academy of Sciences 04/2012; 109(19):7356-61. · 9.68 Impact Factor
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ABSTRACT: 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.
Development 11/2011; 139(1):33-45. · 6.60 Impact Factor
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ABSTRACT: Expression of the basic helix-loop-helix factor Hairy and Enhancer of Split-1 (Hes1) is required for normal development of a number of tissues during embryonic development. Depending on context, Hes1 may act as a Notch signalling effector which promotes the undifferentiated and proliferative state of progenitor cells, but increasing evidence also points to Notch independent regulation of Hes1 expression. Here we use high resolution confocal scanning of EGFP in a novel BAC transgenic mouse reporter line, Tg(Hes1-EGFP)(1Hri), to analyse Hes1 expression from embryonic day 7.0 (e7.0). Our data recapitulates some previous observations on Hes1 expression and suggests new, hitherto unrecognised expression domains including expression in the definitive endoderm at early somite stages before gut tube closure and thus preceding organogenesis. This mouse line will be a valuable tool for studies addressing the role of Hes1 in a number of different research areas including organ specification, development and regeneration.
Gene Expression Patterns 07/2011; 11(7):415-26. · 2.02 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
Diabetes 08/2010; 59(8):1948-56. · 8.29 Impact Factor
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ABSTRACT: Pancreas morphogenesis and cell differentiation are highly conserved among vertebrates during fetal development. The pancreas develops through simple budlike structures on the primitive gut tube to a highly branched organ containing many specialized cell types. This review presents an overview of key molecular components and important signaling sources illustrated by an extensive three-dimensional (3D) imaging of the developing mouse pancreas at single cell resolution. The 3D documentation covers the time window between embryonic days 8.5 and 14.5 in which all the pancreatic cell types become specified and therefore includes gene expression patterns of pancreatic endocrine hormones, exocrine gene products, and essential transcription factors. The 3D perspective provides valuable insight into how a complex organ like the pancreas is formed and a perception of ventral and dorsal pancreatic growth that is otherwise difficult to uncover. We further discuss how this global analysis of the developing pancreas confirms and extends previous studies, and we envisage that this type of analysis can be instrumental for evaluating mutant phenotypes in the future.
Endocrine Reviews 11/2007; 28(6):685-705. · 19.93 Impact Factor
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ABSTRACT: We have developed a wholemount immunofluorescence protocol for the simultaneous detection of up to three proteins in mouse and chicken embryos. Combined with Murray's 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.
Journal of Histochemistry and Cytochemistry 10/2007; 55(9):925-30. · 2.72 Impact Factor
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ABSTRACT: Genetic 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.
In 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.
We 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.
BMC Developmental Biology 02/2007; 7:63. · 2.79 Impact Factor
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ABSTRACT: To understand the molecular mechanisms regulating pancreatic endocrine development and function, pancreatic gene expression was compared between Ngn3-deficient mice and littermate controls on embryonic days 13 and 15. Microarray analysis identified 504 genes with significant differences in expression. Fifty-two of these showed at least twofold reduction in Ngn3 knockouts compared to controls. Many of them were previously described to be involved in endocrine development and function. Among the genes not previously characterized were Rhomboid veinlet-like 4, genes involved in tetrahydrobiopterin biosynthesis and the Iroquois-type homeobox gene Irx1, the latter was selected for further investigation. In situ hybridisation demonstrated that two Iroquois genes, Irx1 and Irx2, were expressed in pancreatic endoderm of wild-type, but not Ngn3 mutant embryos. Furthermore, ectopic Ngn3 induced prominent Irx2 expression in chicken endoderm. Co-labelling established that Irx1 and Irx2 mRNA is located to glucagon-, but not insulin- or somatostatin-producing cells in mice and chicken. These data suggest that Irx1 and Irx2 serve an evolutionary conserved role in the regulation of alpha-cell-specific gene expression.
Journal of Molecular Endocrinology 11/2006; 37(2):301-16. · 3.48 Impact Factor
