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.
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.
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