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First published online June 1, 2005
doi: 10.1242/10.1242/dev.01853

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Notch signaling functions as a binary switch for the determination of glandular and luminal fates of endodermal epithelium during chicken stomach development

¹ Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minamiohsawa, Hachiohji, Tokyo 192-0397, Japan
² Department of Developmental Neurobiology, Graduate School of Medicine, Tohoku University, Sendai, Miyagi 980-8575, Japan
³ Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan

View larger version (142K): [in a new window]   Fig. 1. Expression pattern of genes involved in Notch signaling in early gland formation of PV. Transverse serial sections of stage 28, 29 and 30 were analyzed by in situ hybridization. Throughout this paper, all sections that show normal expression patterns are serial and are obtained from the same sample which is staged exactly. Stage 29 PV show the small glands. For stage 30, boxed areas are shown enlarged in the column on the right. (A-C') Expression of chicken SP and epithelial invagination begins at stage 29. At stage 30, chicken SP-negative cells are still observed among luminal cells (arrows). (D-F') Expression of Smad8 is restricted in invaginating epithelium and begins at stage 29. (G-I') Expression of Delta1 in the epithelium begins at stage 29 in a scattered fashion (arrowheads). (J-L',P-R') Expression of Notch1 and Hairy1 is restricted in uninvaginated epithelium. (M-O',S-U') Expression of Notch2 and Hairy2 is strong in invaginated epithelium. (V-X') Numb is homogeneously expressed in the epithelium. Serial sections made from different stage PV and hybridized with the same probe were placed on the same slide glass to compare intensity of signals. Broken lines in K and Q underline borders between epithelium and mesenchyme. Scale bar: 100 µm.

View larger version (148K): [in a new window]   Fig. 2. Reporter activity in different stages and culture length. pTP-1Venus reporter construct was transfected in stage 28 or 29 PV and cultured for 3, 24 and 48 hours. (A) After 3 hours of culture, stage 29 PV shows the reporter activity in the uninvaginated epithelium in a scattered fashion. No signals were detected in invaginating epithelial cells (arrowheads). (B) Transfected stage 28 PV were cultured for 24 hours. Gland formation is comparable with that in stage 29 PV after 3 hours of culture (A). A significant number of Venus-positive cells (arrowhead) is observed in invaginating epithelial cells. (C) Stage 29 PV were cultured for 48 hours. Scattered Venus-positive cells are still observed, and obvious gland cells show reporter activity. Scale bar: 100 µm.

View larger version (123K): [in a new window]   Fig. 3. Comparison of reporter activity with expression of chicken SP. Stage 29 PV were transfected with pTP-1Venus reporter construct and processed for fluorescent in situ hybridization of chicken SP after 3 hours of culture. The reporter activity is observed in a scattered fashion. Venus expression and chicken SP expression do not overlap. Lower three panels are high-magnification views of the boxed areas in the upper and middle panels. Scale bar: 100 µm.

View larger version (59K): [in a new window]   Fig. 4. Delta1 activates Notch signaling in neighboring epithelial cells. (A-C,C') pTP-1Venus was transfected into stage 29 PV and cultured for 3 hours. Venus expression and expression of Delta1 are adjacent. (C') A high-magnification view of boxed area in C. Outline of cell shapes is shown on the right. (D,E) Stage 28 PV were co-transfected with pTP-1Venus and Delta1 expression vector, and cultured for 12 hours after transfection. Reporter activity was not detected. (F-H) pTP-1Venus was first transfected into stage 28 PV, and Delta1 expression vector was subsequently transfected 10 hours after the first transfection. After an additional 14 hours of culture, the reporter activity and Delta1 expression were detected. Exogenous Delta1 induced activation of the reporter in neighboring cells. Scale bar: 100 µm.

View larger version (67K): [in a new window]   Fig. 5. Forced activation of Notch signaling by CNICC89 transfection. Stage 28 PV were transfected with indicated expression vectors and cultured for 72 hours. Control explants transfected with EGFP form simple glands and express differentiation markers (A-C). Transfection of CNICC89 inhibits gland formation and expression of differentiation-marker genes (D-F). More than 100 explants and more than 40 sections from each explant were investigated. Successfully transfected epithelial cells never expressed differentiation marker genes. Scale bar: 100 µm.

View larger version (113K): [in a new window]   Fig. 6. Induction of glandular cell-specific gene expression by CNIC. Stage 28 PV were transfected with TP-1Venus reporter (control) or CNIC and cultured for 36 hours. Serial sections were made from each explant. Gene expressions were also compared with normally developing PV (right three columns; sections in each column are also serial with Figs 1 and 7). All sections hybridized with the same probe were placed on the same slide glass to compare intensity of signals. Wide and efficient activation of Notch signaling is monitored by co-transfection of Venus reporter construct in the neighboring or next neighboring sections (inset in B and not shown). Reporter activity in control explants is also shown (inset in A; neighbor section of A). (B) CNIC transfection induces Smad8 expression. (G,L,Q,V) Expression of chicken SP and of genes that are specifically downregulated in gland cells is repressed. More than 12 explants were investigated in three experiments. More than 15 sections made from each explant were investigated for each probe. No obvious difference between explants was observed. Scale bar: 100 µm.

View larger version (114K): [in a new window]   Fig. 7. Expression of genes similar to undetermined progenitor cells in CNIC-transfected epithelium. Stage 28 PV were transfected with TP-1Venus reporter (control) or CNIC and cultured for 36 hours. Serial sections were made from the same sample used in Fig. 6. Activation of Notch signaling is monitored by co-transfection of Venus reporter construct in neighboring or next neighboring sections of each panel (insets in B,K; not shown). Sections made from stage 28 of PV and explants, which are hybridized with the same probe, were placed on the same slide glass to compare intensity of signals. Sections of stage 28 PV are also serial with those in Figs 1 and 6. No apparent change was seen in the intensity of gene expression in comparison with stage 28 PV. More than 12 explants were investigated in three experiments. More than 15 sections made from each explant were investigated for each probe. No obvious difference between explants was observed. Scale bar: 100 µm.

View larger version (70K): [in a new window]   Fig. 8. Numb and Su(H)DBM rescued the effect of CNIC. Stage 28 PV, co-transfected with pTP-1Venus reporter construct, CNIC expression vector, and Notch inhibitor construct, were cultured for 36 hours. Both Numb and Su(H)DBM reduce excess activation of Notch signaling (compare B and D with F) and reduce well-spaced gland formation (lack of chicken SP expression in neighboring sections; compare A and C with E). More than 12 explants were investigated. More than 30 sections from each explant were investigated in three experiments. Scale bar: 100 µm.

View larger version (116K): [in a new window]   Fig. 9. Numb and Su(H)DBM transfected cells preferentially contribute to the luminal epithelium. Efficiently transfected Numb (I') and Su(H)DBM (E') suppressed gland formation. Numb-transfected cells (anti-FLAG immunostaining; G',I') and Su(H)DBM-transfected cells (co-transfected EGFP; C',E') selectively localize in the luminal epithelium but not the small glands (C',G'; arrowheads). Expression of EGFP in control explants was observed both in the luminal and glandular epithelium (A-B). Chicken SP expression in the neighboring sections (rightmost column) indicates the differentiation of luminal epithelium. Gland formation was morphologically confirmed (leftmost column). More than 100 explants for Numb and 12 explants for Su(H)DBM were investigated. More than 30 sections from each explant were investigated in at least three experiments. Arrowheads indicate small glands. Scale bar: 100 µm.

View larger version (30K): [in a new window]   Fig. 10. Models of fate determination of the endodermal epithelium and invagination. (A) Notch1 activation instructs endodermal epithelial cells to immature glandular progenitor fate. Remaining epithelial cell takes luminal fate under the influence of luminal inducers. Until Notch1 signaling is inactivated, progenitor glandular cells remain immature. As Notch signaling stops, immature glandular cells undergo gland cell differentiation. (B) Until stage 28, epithelial cells are homogenous. Slightly before stage 29 (stage 29–), when Delta1 expression starts, luminal differentiation and specification of glandular progenitors commence. From stage 29 onwards, as Notch1 signaling is gradually inactivated, glandular progenitors undergo differentiation and invaginate into mesenchyme. Luminal inducer(s) compete with Notch pathway and decrease number of gland progenitor.