- Research article
- Open Access
Preservation of proliferating pancreatic progenitor cells by Delta-Notch signaling in the embryonic chicken pancreas
© Ahnfelt-Rønne et al; licensee BioMed Central Ltd. 2007
- Received: 08 December 2006
- Accepted: 07 June 2007
- Published: 07 June 2007
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.
- Notch Signaling
- Endocrine Cell
- Endocrine Differentiation
- Notch Target Gene
- Hes1 Expression
The pancreas is an organ containing both exocrine and endocrine cell populations. The exocrine pancreas consists of acini and ducts that produce and transport enzymes and bicarbonate to the digestive tract. Lineage tracing studies have revealed that both endocrine and exocrine cells are derived from Pdx1-expressing progenitors [1–3]. The endocrine cells are organized in the islets of Langerhans which contain five distinct cell types, each characterized by the production of specific peptide hormones [4–6]. Endocrine cells begin to appear soon after the first morphological signs of pancreas formation which occurs approximately at the 25-somite stage in the mouse and chicken . Endocrine development depends on Neurogenin3 (Ngn3) and is initiated by the onset of Ngn3 expression in a subset of pancreatic progenitor cells [8–11]. All endocrine cells are derived from Ngn3 expressing precursors and the vast majority, if not all, of the Ngn3 expressing cells are committed to the endocrine lineage [1, 12]
Several studies have demonstrated that Notch signaling is involved in the development of endocrine cells in the pancreas. Mice harboring mutations in the Notch pathway genes Dll1, RBK-Jκ, and Hes1 all display precocious and excessive endocrine development as early as E9.5 [10, 13]. At this stage there is an increase in the numbers of Ngn3 positive cells in Dll1 and RBP-Jκ mutants, and at E10.5 an increase in endocrine cells. Ngn3 expression was not analyzed in Hes1 mutants but these had increased numbers of glucagon-producing cells at E9.5. Together these studies suggest that Notch signaling prevents endocrine differentiation through a mechanism known as lateral inhibition where the Notch ligand Dll1, expressed in differentiating cells, signals through Notch receptors on adjacent cells thereby keeping them undifferentiated or acquiring a secondary fate. However, Notch signaling may act to regulate differentiation by controlling the proneural genes at the transcriptional level or Notch signaling could act in already committed precursors by inhibiting proneural factors posttranslationally. These are not mutually exclusive mechanisms and indeed both have been proposed [14–16]. Later studies have suggested that Notch signaling not only regulates endocrine specification but also inhibits exocrine differentiation [17, 18]. This was further supported by the finding that Hes1, a Notch target gene, is active in exocrine precursors in the mouse pancreas and prevents their terminal differentiation, and that loss of Notch signaling in zebrafish accelerate exocrine differentiation . Subsequently, Jagged mediated Notch signaling has been suggested to mediate a fate choice between exocrine and intrapancreatic duct fate from a common precursor cell in zebrafish . This is most likely different from the situation in mice where duct cell progenitors and exocrine progenitors appear to diverge very early in development between E9.5 and E11.5, prior to expression of Jagged-1 [1, 21].
Here we show that the expression of pro-endocrine bHLH factors and Notch pathway components is conserved in the embryonic chicken pancreas and identify Hes6-1 as being expressed in the endocrine lineage in the embryonic pancreas. We demonstrate that inhibition of Notch signaling results in increased endocrine differentiation, and that activated Notch1 (Notch1ICD) blocks endocrine development and maintains proliferation of pancreatic progenitor cells in the embryonic chicken pancreas. We demonstrate that Notch1ICD is able to inhibit Ngn3 activity as it prevents Ngn3 induced endocrine differentiation, visualized by loss of NeuroD, Myt1, Hes6-1, Pax6, βIII-tubulin, and glucagon expression but, remarkably, without affecting delamination of the Ngn3-expressing cells from the gut epithelium. We also show that NeuroD-induced endocrine development is sensitive to inhibition by Notch. Lastly, we show that forced expression of Myt1 partly restores βIII-tubulin expression in Notch1ICD expressing cells. Together, our results demonstrate that Notch regulation of pancreatic endocrine development is conserved in chicken, and raises a possibility of Notch mediated inhibition of Ngn3 and NeuroD protein activity.
The embryonic chicken pancreas expresses pro-endocrine bHLH genes
Forced expression of Notch ligands induces endocrine differentiation
To test if ectopic expression of the ligands also induced exocrine differentiation we compared the expression of the exo- and endocrine markers amylase and glucagon, respectively, in control and Delta1 electroporated chicken pancreata (Fig 3D,E). GFP-expressing cells were evenly distributed throughout the pancreas epithelium, including exocrine and endocrine cells in the control pancreas. In contrast, the majority of the GFP positive cells expressed glucagon and were excluded from the exocrine cells in Delta electroporated pancreata. Furthermore, the Delta1-expressing pancreata were consistently smaller, suggesting that Delta1 induces precocious endocrine differentiation at the expense of progenitor expansion and later born cell types such as the exocrine.
Constitutively active Notch1 prevents endocrine differentiation and maintains proliferation
Ngn3 induced cell cycle withdrawal and differentiation is antagonized by Notch1ICD
The results described above suggest that Notch mediated control of endocrine pancreas development is conserved in chicken. Previous results obtained in mice have indicated that Notch signaling acts to repress Ngn3 expression and thereby prevents endocrine differentiation [10, 17, 18]. However, it is also possible that Notch signaling may additionally act in cells that already have initiated Ngn3 expression by antagonizing the function of the encoded protein Ngn3 . To test if activated Notch could interfere with the ability of Ngn3 to induce endocrine differentiation we took advantage of the ability of ectopic Ngn3 to induce endocrine differentiation in the chick endoderm . We confirmed that Ngn3 induced endocrine differentiation in the chick pancreas (Fig. 5). Most Ngn3 positive cells were Nkx6.1 negative and Pax6 positive, suggesting they had differentiated into α-cells (Fig. 5A,B). In agreement with this we found that many Ngn3 positive cells expressed glucagon while insulin-expressing cells were rare (Fig. 5C). Mitotic cells were extremely rare among the Ngn3-expressing cells as judged by MPM2 immunoreactivity (Fig. 5D,I) similar to the situation in mouse where early endocrine cells are post mitotic . To test if active Notch signaling can prevent Ngn3-induced endocrine differentiation in the chick pancreas, we co-electroporated HH st. 13–15 embryos with Ngn3 and Notch1ICD expression vectors and assayed endocrine development after 52 hours. In contrast to embryos electroporated with the Ngn3 vector alone we found that most co-electroporated cells maintained Nkx6.1 expression (Fig. 5E) and displayed a high percentage of MPM2 expression (Fig. 5H,I) whereas very few cells expressed Pax6, glucagon or insulin (Fig. 5F,G). This result demonstrates that activated Notch1 can inhibit the function of the Ngn3 protein.
Hes5-1 and Hes6-1 is inversely regulated by Ngn3 and Notch1ICD
Activated Notch1 inhibits Ngn3 and NeuroD induced endocrine differentiation but not delamination
Myt1 can partially antagonize the effects of Notch1ICDon Ngn3 function
The levels of Notch1ICD resulting from forced expression are likely much higher than found during native Notch signaling events. Our experiments therefore do not rule out that Hes6-1 and/or MyT1 could render cells refractive to the action of more physiological levels of Notch as seen in neurogenesis . To test if forced expression of MyT1 at high levels could antagonize Notch1ICD function we electroporated chicken endoderm with constructs encoding Ngn3, Notch1ICD and a FLAG tagged Nzf2b isoform of Myt1. This is one of two Myt1 isoforms resulting from alternative transcriptional start sites reported to be expressed at highest levels in the mouse pancreas. Furthermore, it is the only isoform capable of initiating the endocrine differentiation program in the chicken endoderm . We found that expression of MyT1 led to a partial restoration of neuronal classIII β-tubulin expression (Fig. 8I–K), indicating that some aspects of endocrine differentiation have been rescued in these cells. Expression of Pax6, glucagon and somatostatin was, however, not restored under these conditions (not shown), suggesting that other factors are needed to overcome the repression by Notch1ICD.
The pancreatic expression of bHLH genes and their sensitivity to Notch mediated inhibition observed in this study suggests that endocrine development is highly conserved between mice and chicken.
Overexpression of Notch ligands leads to endocrine differentiation
The observed stimulatory effect of Delta1 over-expression upon endocrine differentiation is in contrast to the effect of Delta1 over-expression in the chicken retina where widespread RCAS-mediated expression of Delta1 inhibits neuronal differentiation , and seemingly at odds with the proposed function of Delta1 as a signal that inhibits endocrine differentiation. However, high level expression of Notch ligands has previously been reported by a number of groups to result in cell autonomous inhibition of Notch signal reception [28–33], raising the possibility that the effect we observe is cell autonomous and the result of inhibition of Notch signaling rather than stimulation of Notch signaling in neighboring cells. In our experiments we used plasmid-based electroporation which results in multiple copies being taken up by the electroporated cells which combined with the very strong promoter used may result in expression levels exceeding those obtained by Henrique and colleagues who used an RCAS retrovirus-mediated delivery . Furthermore, expression of a truncated signaling-defective version of Serrate-1 also resulted in enhanced endocrine development, arguing that the effects we observe with full-length Delta1 and Serrate-1 is the result of a dominant negative effect. This was further corroborated by the ability of Notch1ICD to reverse the effect of the ligands in a cell autonomous manner. However, in spite of the cell-autonomous effect on Notch signal reception, we would expect that cells lying adjacent to cells expressing Delta1 or Serrate1 would be prevented from differentiating through the non-cell autonomous activation of Notch signaling in neighboring cells, while this effect should not be observed for cells lying next to Serrate-d1-expressing cells which are signaling defective. However, since differentiating endocrine cells delaminate from the epithelium we were not able to ascertain whether differentiation of cells initially lying adjacent to Delta1- or Serrate1-expressing cells was delayed or inhibited.
It is noteworthy that we always observe that a fraction of the ligand expressing cells fail to undergo endocrine differentiation and that this fraction increases with time. One possible explanation for this is that these cells are differentiating into acinar cells as premature acinar differentiation is observed in zebrafish with compromised Notch signaling ; however, we found no evidence to suggest that amylase expression occurred prematurely. It is also possible that such cells are committed to a non-endocrine fate and that inhibition of Notch signaling therefore cannot induce endocrine differentiation. In this regard it is notable that forced expression of Ngn3 also fails to induce endocrine differentiation of all the cells that express the introduced gene. However, a trivial explanation may be that the cells that fail to differentiate express the introduced genes at levels that are incompatible with endocrine differentiation.
The endocrinogenic effect of Notch ligand expression was confined to the pancreatic endoderm. In spite of widespread expression in prospective duodenum we found no signs of endocrine development. This is most likely due to the restriction of Ngn3 expression to the pancreas anlage at this stage, but apparently contrasts with recent data from Gu and colleagues who found that forced expression of Manic Fringe in prospective duodenum led to endocrine development through inhibition of Notch signaling . The reason for this discrepancy is unclear at present but could be related to the different mechanisms by which Manic Fringe and high levels of Delta1 inhibits Notch signal reception.
Activated Notch inhibits endocrine promoting Ngn3 activity
Previous studies of Notch function in pancreatic endocrine development have not considered a potential role for Notch mediated inhibition of Ngn3 and/or NeuroD protein function. Several examples of post-translational inhibition of pro-neural function by Notch have been described in the literature. The function of the exocrine bHLH factor p48-PTF1 has been shown to be inhibited via heterodimerization to Hes1 . In Drosophila, several Notch target genes of the E(Spl) class have been shown to dimerize with pro-neural bHLH factors and antagonize the function of these , in Xenopus X-NGN1 function can be antagonized by activated Notch1 [39, 40], and in human cell lines Notch signaling has been demonstrated to lead to a rapid degradation of the human achaete-scute homolog hASH1 . It is possible that similar mechanisms regulate Ngn3 function. It is also possible that regulatory sequences of genes induced by Ngn3 also contain Hes binding sites and that Hes repressor activity is dominant over Ngn3. Although our results do not prove that Notch signaling is active in Ngn3-expressing precursors they do raise the possibility that Notch might have a second effect in endocrine differentiation in that it could regulate the levels and duration of Ngn3 activity. A similar but not identical experiment has been conducted by Murtaugh and colleagues who used Ngn3-Cre mice to conditionally mix-express Notch1ICD in endocrine progenitor cells which led to inhibition of endocrine differentiation . However, since Ngn3 expression was not investigated in that study it is unclear whether this block in endocrine development was caused by inhibition of Ngn3 gene expression or Ngn3 protein activity. Recently, Notch1ICD was shown to re-direct endocrine progenitors to a duct cell fate when expression was induced in Pax4-expressing cells . Presumably, this effect would require inhibition of Ngn3 as Pax4 is a target of Ngn3.
A surprising finding is that activated Notch did not result in significant upregulation of Hes1 in the pancreas but rather appeared to repress the expression of this Notch effector gene. As discussed above this may be a secondary effect to the induction of Hes5-1. In a transgenic mouse expressing Notch1ICD under the Pdx1 promoter, Hes1 protein could not be detected in most of the Notch1ICD expressing pancreatic cells even though Ngn3 expression was almost completely abolished throughout the epithelium. It was speculated that the Hes1 protein is expressed at levels too low for detection by immunohistochemistry. An alternative interpretation is that the effect of Notch1ICD is mediated by other Hes factors such as Hes5, or that Notch signaling may be required but not sufficient for Hes1 expression under normal circumstances.
Surprisingly, Ngn3-induced delamination still occurred in the presence of Notch1ICD. The molecular mechanism controlling delamination of endocrine cells is poorly defined but it is possible that Ngn3-targets responsible for delamination are less sensitive to Notch-mediated repression than targets genes involved in endocrine differentiation, e.g., if genes required for delamination lack Hes binding sites in their regulatory elements. Intriguingly, differentiation and delamination is also uncoupled in other circumstances. A mutant Ngn3 protein carrying a missense mutation (R107S) is hypomorphic in regard to endocrine-inducing activity, but still capable of inducing delamination to the same extent as wild-type Ngn3 .
Activated Notch antagonize Ngn3 as well as NeuroD function
Conditional expression of Notch1ICD in mature insulin-producing beta cells has no apparent effect on the beta cell phenotype . Since NeuroD function is required for beta cell survival and function [49, 50] we speculated that NeuroD might be refractive to the effects of activated Notch. However, endocrine differentiation induced by forced NeuroD expression in chicken endoderm was completely prevented by co-expression of Notch1ICD. This could reflect a mechanistic change in NeuroD function in mature endocrine cells compared to precursor cells. In agreement with this notion we found that a few endocrine cells in the pancreas expressed Notch1ICD probably reflecting the occasional transfection of existing endocrine cells at the time of electroporation. Alternatively, it is possible that the ROSA26 locus, used to conditionally express Notch1ICD by Murtaugh and colleagues, results in appreciably lower levels of expression than we obtain by electroporation and that high level expression of Notch1ICD is required for repression of NeuroD function. NeuroD is also capable of inducing neurogenesis when expressed in Xenopus ectoderm [39, 51] but it is unclear if activated Notch can inhibit this function of NeuroD. In one study, X-NGNR-1 and XNeuroD were equally sensitive to inhibition by X-NotchICD in side-by-side comparisons , while in another study the number of neurons formed in response to X-NGNR-1 was reduced by X-NotchICDwhich was not the case for XNeuroD induced neurons . A number of observations support a similar function in pancreatic endocrine development; forced expression of either Ngn3 or NeuroD in the pancreas can induce the endocrine differentiation program [8, 10, 11] and Ngn3 induces NeuroD expression [10, 11], while NeuroD cannot induce Ngn3 expression (this study) suggesting that a unidirectional bHLH cascade may also regulate endocrine determination and differentiation.
MyT1 antagonism of Notch1ICDactivity
During Xenopus neurogenesis MyT1 can in cooperation with X-NGNR-1 overcome the repressive activity of Notch upon X-NGNR-1 . We observe only a partial antagonistic effect of MyT1 on Notch mediated repression of Ngn3 function. The reasons for this incomplete rescue is unclear but it is possible that the chicken electroporation system results in far higher levels of activated Notch than seen with mRNA injection in Xenopus oocytes. It is possible that MyT1 would have a greater effect if more physiological expression levels were used. Nevertheless, the expression of known Notch signaling modifiers such as Hes6-1, Myt1, and Manic Fringe in endocrine precursors [44, 46] suggests that Notch signaling is finely regulated during the early stages of endocrine differentiation. However, conditional loss-of-function studies in mice are needed to further elucidate the cellular and biochemical mechanisms of Notch signaling during these early stages of endocrine development.
Together the data presented here establish that pancreatic endocrine development in the chicken relies in a conserved bHLH cascade under inhibitory control of Notch signaling. While this may not be surprising it does lay the ground for further studies that take advantage of the ease at which chicken embryos can be manipulated.
Our results also raise the possibility that Notch could act in Ngn3-expressing cells and delay their differentiation. To determine whether Notch in fact does act upon Ngn3 in vivo will require sophisticated 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.
All expression constructs were made in a pCIG vector  optimized for EGFP intensity (pCIG5 or pCGIG5 with a Gateway (invitrogen) recombination cassette, kindly provided by Anne Grapin-Botton). A pcDNAI vector containing a cDNA encoding N-terminal haemaglutinin (HA) tagged mouse Ngn3 was kindly provided by François Guillemot. This vector was cut with HindIII and filled with klenow. The cDNA was released with NotI and the entire fragment was cloned into the EcoRV/NotI sites of pCIG5. The cDNA encoding N-terminally HA tagged rat NeuroD has been described previously . The coding sequence was amplified by PCR with the primers CACCATGGGCTACCCATAGAT and CTAATCGTGAAAGATGGCATT and TOPO cloned into the pENTR/D vector (Invitrogen). Subsequently, HA-NeuroD was cloned by recombination into pCGIG5. The cDNA encoding C-terminally myc tagged rat Notch1ICD has been described previously . An EcoRI fragment containing the entire coding sequence was cloned into the EcoRI site of pCIG5. The mouse Myt1-7zf gene was FLAG tagged (MDYKDDDDKMTKSYSESGL) at the N terminus using PCR and cloned into pENTR/D (invitrogen). The tagged version of Myt1-7zf was cloned into the pCGIG5 vector using gateway cloning. The cDNAs encoding FLAG tagged chicken Delta1 and HA tagged chicken Serrate1 and the dominant negative Serrate1-d1 were a kind gift from Ken-ichi Katsube . Delta1-FLAG was released with ApaI (Klenow blunted) and BamHI and ligated to EcoRI (Klenow blunted) and BamHI sites in pEntr 2B (Invitrogen). The coding sequence was cloned into the pCGIG vector by Gateway recombination. The Serrate constructs were cloned into XhoI and EcoRI sites of pEntr 2B and subsequently inserted in the pCGIG vector by Gateway cloning. All constructs were verified by sequencing.
In ovo endoderm electroporation
Electroporations were performed as described in . Briefly, fertilized White Leghorn eggs (Triova, Denmark) were incubated at 38°C, 50% relative humidity. Electroporation was performed on chicken embryos at HH st. 10–15  : The eggs were windowed and a solution of DNA (with a fixed concentration of 2 μg/μl in PBS w/o Ca2+ and Mg2+, 1 mM MgCl2, 3 mg/ml carboxymethylcellulose, 0.66 mg/ml Fast Green) was injected into the blastocoel. An anode was placed over and a cathode under the embryo along the anteroposterior axis of the embryo using a micromanipulator. Depending on stage, three to five square 50 ms pulses of 7–15 V were applied from a BTX ECM830 electroporator and the eggs were sealed and returned to the incubator and allowed to develop for 52–72 hours. Electroporated embryos were immunostained with an antibody specific for the epitope tags and co-localization with GFP was assayed: As expected we found that most GFP positive cells also expressed the epitope tags (not shown) and we therefore used GFP as a measure of productive expression. For co-expression experiments double immunohistochemistry against the different epitope tags was used to verify co-expression (not shown).
Immunohistochemistry and statistical analyses
Whole mount immunofluorescent stainings: The stomach with the proximal duodenum along with the pancreas was dissected from the embryos and fixed over night in 4% PFA at 4°C. Specimens were processed as in . For IHC on sections, embryos were fixed in 4% PFA at 4°C over night, equilibrated in 30% sucrose in PBS and frozen in Tissue-Tek OCT compound (Sakura finetek). 10 μm sections were obtained on a Leica CM3050 S cryostat and collected on SuperFrost plus (Menzel-Gläser) slides. Sections were washed in PBS and blocked for at least 1 hour in 0.5% TNB (PerkinElmer). Primary antibodies were diluted in 0.5% TNB and applied over night. Secondary antibodies were applied for 1 hour after several washes in PBS and slides were mounted in 20% glycerol (in 50 mM Tris buffer adjusted to pH8.4). Primary antibodies were: Mouse-anti-HA (HA.11, Babco), rabbbit-anti-myc (A14, Santa Cruz), mouse-anti-FLAG (M2, Sigma), Mouse-anti-glucagon (Glu-001, Novo Nordisk A/S), guinea pig-anti-insulin (ab7842, Abcam), rabbit-anti-somatostatin (A566, DAKO), rabbit-anti-amylase (A 8273, Sigma), rabbit-anti-Laminin (L9393, Sigma), mouse-anti-MPM2 (M3514, DAKO), rabbit-anti-βIII-Tubulin (PRB-435P, Nordic Biosite). rabbit-anti-Nkx6.1 , rabbit-anti-GFP (632460, Clontech), (DSHB) The mouse anti-Pax6 monoclonal antibody developed by Atsushi Kawakami was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Secondary antibodies were purchased from Jackson ImmunoResearch (Cy3 conjugated donkey-anti-rabbit and donkey-anti-guinea pig and Cy5 conjugated donkey-anti-mouse). Negative controls include omission of primary antibodies and staining of non-electroporated embryos Images were collected by confocal microscopy on an LSM 510 META Laser Scanning Microscope (Carl Zeiss) and fluorescent signals were assigned false colors with Zeiss LSM software. Quantification was performed by sampling sections covering all the electroporated endoderm (between 7 and 10 sections from each embryo) and counting at least between 350 and 750 GFP+ cells from embryos electroporated at HH st. 10–12 and between 1200 and 4800 for embryos electroporated at HH st. 13–15. Between 3 and 13 embryos were analyzed quantitatively for each condition. Statistical analyses were performed using Student's T-test.
In situ hybridization
Digoxigenin labelled cRNA probes were generated by in vitro transcription using reagents from Roche according to manufacturer's instruction. The following chicken cDNA clones were used for probe generation: Ngn3 was kindly provided by A. Grapin-Botton and J. M. Matter. Delta1, Notch1, Serrate1 and Serrate2 were generous gifts from D. Henrique [56, 57]. NeuroD: ChEST695k11, Hes1: ChEST900h16, Hes6-1: ChEST296l7, Hes6-2: ChEST529e6, Hes5-1: ChEST295o19, Myt1: ChEST672b12, Notch2: ChEST909l22. ChEST clones were from the BBSRC Chick EST database and purchased from MRC gene service. Wholemount ISH was performed essentially as in . ISH on frozen sections: Frozen sections were thawed and denatured cRNA probes diluted to 1 ng/μl in hybridization solution (50% formamide, 1x salt, 10% Dextran sulphate, 1 mg/ml yeast tRNA, 1x Denhardts) were added directly to the sections. The sections were covered with a cover slip and placed in a humidified chamber (humidified with 5×SCC, 50% formamide) at 65°C over night. The slides were washed 3 times in wash solution (50% formamide, 1× SCC, 0.1% Tween-20) at 65°C followed by several washes in MABT (100 mM maleic acid, 150 mM NaCl, 0.1% Tween-20, adjusted to pH7.5 with NaOH) at room temperature. The sections were blocked in 2% blocking reagent (Roche), 20% heat inactivated goat serum in MABT for 1 hour and incubated over night with an AP conjugated sheep-anti-DIG (Roche) (1:2500 in blocking solution). The sections were washed in MABT and then equilibrated in NTMT (50 mM Tris, 0.1 M NaCl, 0.02 M MgCl2, 0.1% Tween-20, pH9.5). AP activity was visualized with NBT/BCIP in NTMT. Images were collected with a Hamamatsu C5810 cooled CCD camera mounted on an Olympus BX51 microscope and processed in Adobe Photoshop™.
We are grateful to Louise C. Rosenberg for technical assistance and to Anne Grapin-Botton, Jean-Marc Matter, Domingos Henrique, Francois Guillemot, and Ken-ichi Katsube for reagents. P.S. was supported by JDRF and EU 6th Framework Program.
- Gu G, Dubauskaite J, Melton DA: Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development. 2002, 129: 2447-2457.PubMedGoogle Scholar
- Gu G, Brown JR, Melton DA: Direct lineage tracing reveals the ontogeny of pancreatic cell fates during mouse embryogenesis. Mech Dev. 2003, 120: 35-43. 10.1016/S0925-4773(02)00330-1.View ArticlePubMedGoogle Scholar
- Herrera PL: Adult insulin- and glucagon-producing cells differentiate from two independent cell lineages. Development. 2000, 127: 2317-2322.PubMedGoogle Scholar
- Slack JM: Developmental biology of the pancreas. Development. 1995, 121: 1569-1580.PubMedGoogle Scholar
- Prado CL, Pugh-Bernard AE, Elghazi L, Sosa-Pineda B, Sussel L: Ghrelin cells replace insulin-producing beta cells in two mouse models of pancreas development. Proc Natl Acad Sci U S A. 2004, 101: 2924-2929. 10.1073/pnas.0308604100.PubMed CentralView ArticlePubMedGoogle Scholar
- Heller RS, Jenny M, Collombat P, Mansouri A, Tomasetto C, Madsen OD, Mellitzer G, Gradwohl G, Serup P: Genetic determinants of pancreatic epsilon-cell development. Dev Biol. 2005, 286: 217-224. 10.1016/j.ydbio.2005.06.041.View ArticlePubMedGoogle Scholar
- Kim SK, Hebrok M, Melton DA: Pancreas development in the chick embryo. Cold Spring Harb Symp Quant Biol. 1997, 62: 377-383.View ArticlePubMedGoogle Scholar
- Schwitzgebel VM, Scheel DW, Conners JR, Kalamaras J, Lee JE, Anderson DJ, Sussel L, Johnson JD, German MS: Expression of neurogenin3 reveals an islet cell precursor population in the pancreas. Development. 2000, 127: 3533-3542.PubMedGoogle Scholar
- Gradwohl G, Dierich A, LeMeur M, Guillemot F: neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc Natl Acad Sci U S A. 2000, 97: 1607-1611. 10.1073/pnas.97.4.1607.PubMed CentralView ArticlePubMedGoogle Scholar
- Apelqvist A, Li H, Sommer L, Beatus P, Anderson DJ, Honjo T, Hrabe de Angelis M, Lendahl U, Edlund H: Notch signalling controls pancreatic cell differentiation. Nature. 1999, 400: 877-881. 10.1038/23716.View ArticlePubMedGoogle Scholar
- Grapin-Botton A, Majithia AR, Melton DA: Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes. Genes Dev. 2001, 15: 444-454. 10.1101/gad.846001.PubMed CentralView ArticlePubMedGoogle Scholar
- Schonhoff SE, Giel-Moloney M, Leiter AB: Neurogenin 3-expressing progenitor cells in the gastrointestinal tract differentiate into both endocrine and non-endocrine cell types. Dev Biol. 2004, 270: 443-454. 10.1016/j.ydbio.2004.03.013.View ArticlePubMedGoogle Scholar
- Jensen J, Pedersen EE, Galante P, Hald J, Heller RS, Ishibashi M, Kageyama R, Guillemot F, Serup P, Madsen OD: Control of endodermal endocrine development by Hes-1. Nat Genet. 2000, 24: 36-44. 10.1038/72814.View ArticlePubMedGoogle Scholar
- Alifragis P, Poortinga G, Parkhurst SM, Delidakis C: A network of interacting transcriptional regulators involved in Drosophila neural fate specification revealed by the yeast two-hybrid system. Proc Natl Acad Sci U S A. 1997, 94: 13099-13104. 10.1073/pnas.94.24.13099.PubMed CentralView ArticlePubMedGoogle Scholar
- Sriuranpong V, Borges MW, Strock CL, Nakakura EK, Watkins DN, Blaumueller CM, Nelkin BD, Ball DW: Notch signaling induces rapid degradation of achaete-scute homolog 1. Mol Cell Biol. 2002, 22: 3129-3139. 10.1128/MCB.22.9.3129-3139.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Skipper M, Lewis J: Getting to the guts of enteroendocrine differentiation. Nat Genet. 2000, 24: 3-4. 10.1038/71653.View ArticlePubMedGoogle Scholar
- Hald J, Hjorth JP, German MS, Madsen OD, Serup P, Jensen J: Activated Notch1 prevents differentiation of pancreatic acinar cells and attenuate endocrine development. Dev Biol. 2003, 260: 426-437. 10.1016/S0012-1606(03)00326-9.View ArticlePubMedGoogle Scholar
- Murtaugh LC, Stanger BZ, Kwan KM, Melton DA: Notch signaling controls multiple steps of pancreatic differentiation. Proc Natl Acad Sci U S A. 2003, 100: 14920-14925. 10.1073/pnas.2436557100.PubMed CentralView ArticlePubMedGoogle Scholar
- Esni F, Ghosh B, Biankin AV, Lin JW, Albert MA, Yu X, MacDonald RJ, Civin CI, Real FX, Pack MA, Ball DW, Leach SD: Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas. Development. 2004, 131: 4213-4224. 10.1242/dev.01280.View ArticlePubMedGoogle Scholar
- Yee NS, Lorent K, Pack M: Exocrine pancreas development in zebrafish. Dev Biol. 2005, 284: 84-101. 10.1016/j.ydbio.2005.04.035.View ArticlePubMedGoogle Scholar
- Lammert E, Brown J, Melton DA: Notch gene expression during pancreatic organogenesis. Mech Dev. 2000, 94: 199-203. 10.1016/S0925-4773(00)00317-8.View ArticlePubMedGoogle Scholar
- Jensen J, Heller RS, Funder-Nielsen T, Pedersen EE, Lindsell C, Weinmaster G, Madsen OD, Serup P: Independent development of pancreatic alpha- and beta-cells from neurogenin3-expressing precursors: a role for the notch pathway in repression of premature differentiation. Diabetes. 2000, 49: 163-176. 10.2337/diabetes.49.2.163.View ArticlePubMedGoogle Scholar
- Hamburger VH: A series of normal stages in the development of the chick embryo. J Exp Morphol. 1951, 88: 49-92. 10.1002/jmor.1050880104.View ArticleGoogle Scholar
- Palmeirim I, Henrique D, Ish-Horowicz D, Pourquie O: Avian hairy gene expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis. Cell. 1997, 91: 639-648. 10.1016/S0092-8674(00)80451-1.View ArticlePubMedGoogle Scholar
- Sander M, Sussel L, Conners J, Scheel D, Kalamaras J, Dela Cruz F, Schwitzgebel V, Hayes-Jordan A, German M: Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas. Development. 2000, 127: 5533-5540.PubMedGoogle Scholar
- Pedersen JK, Nelson SB, Jorgensen MC, Henseleit KD, Fujitani Y, Wright CV, Sander M, Serup P: Endodermal expression of Nkx6 genes depends differentially on Pdx1. Dev Biol. 2005, 288: 487-501. 10.1016/j.ydbio.2005.10.001.View ArticlePubMedGoogle Scholar
- Fior R, Henrique D: A novel hes5/hes6 circuitry of negative regulation controls Notch activity during neurogenesis. Dev Biol. 2005, 281: 318-333. 10.1016/j.ydbio.2005.03.017.View ArticlePubMedGoogle Scholar
- Sakamoto K, Ohara O, Takagi M, Takeda S, Katsube K: Intracellular cell-autonomous association of Notch and its ligands: a novel mechanism of Notch signal modification. Dev Biol. 2002, 241: 313-326. 10.1006/dbio.2001.0517.View ArticlePubMedGoogle Scholar
- Sun X, Artavanis-Tsakonas S: The intracellular deletions of Delta and Serrate define dominant negative forms of the Drosophila Notch ligands. Development. 1996, 122: 2465-2474.PubMedGoogle Scholar
- Jacobsen TL, Brennan K, Arias AM, Muskavitch MA: Cis-interactions between Delta and Notch modulate neurogenic signalling in Drosophila. Development. 1998, 125: 4531-4540.PubMedGoogle Scholar
- Klein T, Brennan K, Arias AM: An intrinsic dominant negative activity of serrate that is modulated during wing development in Drosophila. Dev Biol. 1997, 189: 123-134. 10.1006/dbio.1997.8564.View ArticlePubMedGoogle Scholar
- Franklin JL, Berechid BE, Cutting FB, Presente A, Chambers CB, Foltz DR, Ferreira A, Nye JS: Autonomous and non-autonomous regulation of mammalian neurite development by Notch1 and Delta1. Curr Biol. 1999, 9: 1448-1457. 10.1016/S0960-9822(00)80114-1.View ArticlePubMedGoogle Scholar
- Micchelli CA, Rulifson EJ, Blair SS: The function and regulation of cut expression on the wing margin of Drosophila: Notch, Wingless and a dominant negative role for Delta and Serrate. Development. 1997, 124: 1485-1495.PubMedGoogle Scholar
- Davis FM, Tsao TY, Fowler SK, Rao PN: Monoclonal antibodies to mitotic cells. Proc Natl Acad Sci U S A. 1983, 80: 2926-2930. 10.1073/pnas.80.10.2926.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhu X, Zhang J, Tollkuhn J, Ohsawa R, Bresnick EH, Guillemot F, Kageyama R, Rosenfeld MG: Sustained Notch signaling in progenitors is required for sequential emergence of distinct cell lineages during organogenesis. Genes Dev. 2006, 20: 2739-2753. 10.1101/gad.1444706.PubMed CentralView ArticlePubMedGoogle Scholar
- Yoon K, Gaiano N: Notch signaling in the mammalian central nervous system: insights from mouse mutants. Nat Neurosci. 2005, 8: 709-715. 10.1038/nn1475.View ArticlePubMedGoogle Scholar
- Hatakeyama J, Bessho Y, Katoh K, Ookawara S, Fujioka M, Guillemot F, Kageyama R: Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation. Development. 2004, 131: 5539-5550. 10.1242/dev.01436.View ArticlePubMedGoogle Scholar
- Matsuda Y, Wakamatsu Y, Kohyama J, Okano H, Fukuda K, Yasugi S: Notch signaling functions as a binary switch for the determination of glandular and luminal fates of endodermal epithelium during chicken stomach development. Development. 2005, 132: 2783-2793. 10.1242/dev.01853.View ArticlePubMedGoogle Scholar
- Ma Q, Kintner C, Anderson DJ: Identification of neurogenin, a vertebrate neuronal determination gene. Cell. 1996, 87: 43-52. 10.1016/S0092-8674(00)81321-5.View ArticlePubMedGoogle Scholar
- Olson EC, Schinder AF, Dantzker JL, Marcus EA, Spitzer NC, Harris WA: Properties of ectopic neurons induced by Xenopus neurogenin1 misexpression. Mol Cell Neurosci. 1998, 12: 281-299. 10.1006/mcne.1998.0712.View ArticlePubMedGoogle Scholar
- Pedersen AH, Heller RS: A possible role for the canonical Wnt pathway in endocrine cell development in chicks. Biochem Biophys Res Commun. 2005, 333: 961-968. 10.1016/j.bbrc.2005.05.189.View ArticlePubMedGoogle Scholar
- Bae S, Bessho Y, Hojo M, Kageyama R: The bHLH gene Hes6, an inhibitor of Hes1, promotes neuronal differentiation. Development. 2000, 127: 2933-2943.PubMedGoogle Scholar
- Bellefroid EJ, Bourguignon C, Hollemann T, Ma Q, Anderson DJ, Kintner C, Pieler T: X-MyT1, a Xenopus C2HC-type zinc finger protein with a regulatory function in neuronal differentiation. Cell. 1996, 87: 1191-1202. 10.1016/S0092-8674(00)81815-2.View ArticlePubMedGoogle Scholar
- Gu G, Wells JM, Dombkowski D, Preffer F, Aronow B, Melton DA: Global expression analysis of gene regulatory pathways during endocrine pancreatic development. Development. 2004, 131: 165-179. 10.1242/dev.00921.View ArticlePubMedGoogle Scholar
- Henrique D, Hirsinger E, Adam J, Le Roux I, Pourquie O, Ish-Horowicz D, Lewis J: Maintenance of neuroepithelial progenitor cells by Delta-Notch signalling in the embryonic chick retina. Curr Biol. 1997, 7: 661-670. 10.1016/S0960-9822(06)00293-4.View ArticlePubMedGoogle Scholar
- Xu Y, Wang S, Zhang J, Zhao A, Stanger BZ, Gu G: The fringe molecules induce endocrine differentiation in embryonic endoderm by activating cMyt1/cMyt3. Dev Biol. 2006, 297: 340-349. 10.1016/j.ydbio.2006.04.456.View ArticlePubMedGoogle Scholar
- Greenwood AL, Li S, Jones K, Melton DA: Notch signaling reveals developmental plasticity of Pax4(+) pancreatic endocrine progenitors and shunts them to a duct fate. Mech Dev. 2007, 124: 97-107. 10.1016/j.mod.2006.11.002.View ArticlePubMedGoogle Scholar
- Jensen JN, Rosenberg LC, Hecksher-Sorensen J, Serup P: Mutant neurogenin-3 in congenital malabsorptive diarrhea. N Engl J Med. 2007, 356: 1781-1782. 10.1056/NEJMc063247.View ArticlePubMedGoogle Scholar
- Malecki MT, Jhala US, Antonellis A, Fields L, Doria A, Orban T, Saad M, Warram JH, Montminy M, Krolewski AS: Mutations in NEUROD1 are associated with the development of type 2 diabetes mellitus. Nat Genet. 1999, 23: 323-328. 10.1038/15500.View ArticlePubMedGoogle Scholar
- Naya FJ, Huang HP, Qiu Y, Mutoh H, DeMayo FJ, Leiter AB, Tsai MJ: Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neuroD-deficient mice. Genes Dev. 1997, 11: 2323-2334.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee JE, Hollenberg SM, Snider L, Turner DL, Lipnick N, Weintraub H: Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. Science. 1995, 268: 836-844. 10.1126/science.7754368.View ArticlePubMedGoogle Scholar
- Megason SG, McMahon AP: A mitogen gradient of dorsal midline Wnts organizes growth in the CNS. Development. 2002, 129: 2087-2098.PubMedGoogle Scholar
- Petersen HV, Jensen JN, Stein R, Serup P: Glucose induced MAPK signalling influences NeuroD1-mediated activation and nuclear localization. FEBS Lett. 2002, 528: 241-245. 10.1016/S0014-5793(02)03318-5.View ArticlePubMedGoogle Scholar
- Ahnfelt-Rønne J, Jørgensen M, Hald J, Madsen O, Serup P, Hecksher-Sørensen J: An improved method for 3D reconstruction of protein expression patterns in intact mouse and chicken embryos and organs. Journal of Histochemistry and Cytochemistry. 2007, in press:Google Scholar
- Jensen J, Serup P, Karlsen C, Nielsen TF, Madsen OD: mRNA profiling of rat islet tumors reveals nkx 6.1 as a beta-cell-specific homeodomain transcription factor. J Biol Chem. 1996, 271: 18749-18758. 10.1074/jbc.271.31.18749.View ArticlePubMedGoogle Scholar
- Myat A, Henrique D, Ish-Horowicz D, Lewis J: A chick homologue of Serrate and its relationship with Notch and Delta homologues during central neurogenesis. Dev Biol. 1996, 174: 233-247. 10.1006/dbio.1996.0069.View ArticlePubMedGoogle Scholar
- Henrique D, Adam J, Myat A, Chitnis A, Lewis J, Ish-Horowicz D: Expression of a Delta homologue in prospective neurons in the chick. Nature. 1995, 375: 787-790. 10.1038/375787a0.View ArticlePubMedGoogle Scholar
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