The tetraspanin Tm4sf3 is localized to the ventral pancreas and regulates fusion of the dorsal and ventral pancreatic buds.
During embryogenesis, the pancreas develops from separate dorsal and ventral buds, which fuse to form the mature pancreas. Little is known about the functional differences between these two buds or the relative contribution of cells derived from each region to the pancreas after fusion. To follow the fate of dorsal or ventral bud derived cells in the pancreas after fusion, we produced chimeric Elas-GFP transgenic/wild-type embryos in which either dorsal or ventral pancreatic bud cells expressed GFP. We found that ventral pancreatic cells migrate extensively into the dorsal pancreas after fusion, whereas the converse does not occur. Moreover, we found that annular pancreatic tissue is composed exclusively of ventral pancreas-derived cells. To identify ventral pancreas-specific genes that may play a role in pancreatic bud fusion, we isolated individual dorsal and ventral pancreatic buds, prior to fusion, from NF38/39 Xenopus laevis tadpoles and compared their gene expression profiles (NF refers to the specific stage of Xenopus development). As a result of this screen, we have identified several new ventral pancreas-specific genes, all of which are expressed in the same location within the ventral pancreas at the junction where the two ventral pancreatic buds fuse. Morpholino-mediated knockdown of one of these ventral-specific genes, transmembrane 4 superfamily member 3 (tm4sf3), inhibited dorsal-ventral pancreatic bud fusion, as well as acinar cell differentiation. Conversely, overexpression of tm4sf3 promoted development of annular pancreas. Our results are the first to define molecular and behavioral differences between the dorsal and ventral pancreas, and suggest an unexpected role for the ventral pancreas in pancreatic bud fusion.
PubMed ID: 19403659
PMC ID: PMC2680106
Article link: Development.
Grant support: DK077197 NIDDK NIH HHS , GM075018 NIGMS NIH HHS , R01 DK077197-04 NIDDK NIH HHS , R01 DK077197 NIDDK NIH HHS
Genes referenced: agr2 celf3 fubp1 gcg hhex ins insm1 neurod1 nkx2-2 pax6 pdia2 prkca ptf1a scg3 sfrp5 tspan8 unnamed
Morpholinos referenced: tspan8 MO1 tspan8 MO2
Article Images: [+] show captions
|Fig. 5. Expression of tm4sf3 in the gastrointestinal tract and pancreas. (A) NF40 whole gut showing localized expression of tm4sf3 to the developing stomach and duodenum. (B) NF44 whole gut. tm4sf3 expression persists in the stomach and duodenum, and is also now detected in the bile duct (bd). (C) Isolated pancreas/liver tissue at NF40, revealing expression of tm4sf3 in the ventral pancreas (vp). Expression is localized to the junction where the two ventral pancreatic buds fused. A Small amount of expression is also evident in the bile duct. li, liver. (D) By NF44, ventral pancreas-specific expression decreased, but increased in the bile duct.|
|Fig. 6. Tm4sf3 is required for acinar and stomach/duodenum development. (A,B) Expression of the general pancreas marker Ptf1a is normal in tm4sf3 morphants (n=7), but also reveals that the dorsal (dp) and ventral (vp) pancreatic buds have not fused. The liver position (L) has changed compared with normal and is present below the pancreatic buds in the region of the stomach/duodenum. (C,D) Expression of the liver marker Hex was normal (n=18). P, pancreas. (E,F) Expression of the stomach/duodenum marker frp5 was almost completely abolished (40/44). (G,H) Schematic highlighting the phenotype seen in Tm4sf3 knockdown embryos. The pancreas normally grows behind the duodenum, as illustrated by the darker shading in the wild-type gut. In tm4sf3 morphants, the dorsal and ventral pancreatic buds do not fuse. (I,J) The acinar cell marker elastase was substantially reduced or completely abolished in Tm4sf3 knockdown embryos (19/27 absent, 8/27 reduced). (K,L) Expression of the early acinar differentiation marker XPDIp was normal in the dorsal and ventral pancreatic buds in tm4sf3 morphants (n=42). (M,N) Insulin expression was normal in the dorsal pancreas in a little over half the cases (16/29). Interestingly, ectopic insulin expression was found in the ventral pancreatic bud in 45% of our samples (13/29). (O,P) No expression of glucagon or somatostatin was detected in the stomach/duodenum (n=8). At this stage, neither is yet expressed in the pancreas. (Q-V) Representative serial sections from an individual isolated NF42 whole gut that were previously stained for ptf1a expression. (W,X) Three-dimensional reconstruction of the samples based on all serial sections. Pancreas is blue, liver is pink, intestine is yellow and the gall bladder is green.|
|Fig. 7. tm4sf3 mRNA rescues the morpholino knockdown phenotype. (A) Control whole gut stained for elastase expression. (B) Whole gut from embryo injected with 20 ng of morpholino. No elastase expression is detected, and the two buds did not fuse. Phenotype seen in 50/58 injected embryos. dp, dorsal pancreas; vp, ventral pancreas. The liver is outlined with a broken red line. (C) Whole gut from embryo co-injected with 20 ng tm4sf3 morpholino and 1800 pg tm4sf3 mRNA. Elastase expression is restored and the dorsal and ventral buds have fused. Rescue was seen in 59/112 injected embryos (53% rescue), 47/112 had the knockdown phenotype (42%) and 6/112 developed an annular pancreas (5%).|
|Fig. 8. Overexpression of tm4sf3 mRNA resulted in annular pancreas development. (A,B) Expression of the acinar cell maker elastase is detected in the ectopic pancreatic tissue (n=19). (D,E) The endocrine marker insulin is not expressed (n=24). (G,H) Development of the liver was not affected by tm4sf3 overexpression (n=17). The annular pancreas is outlined. (J,K) Schematic illustrating the development of annular pancreas. Pink, liver; blue, pancreas; yellow, GI tract. The pancreas normally grows behind the stomach, but in tm4sf3-injected embryos the pancreas also grows around the front of the duodenum. Lighter shading behind the yellow indicates the pancreas. (C,F,I) Representative serial sections from tm4sf3-injected whole gut. The pancreas encircles the stomach completely (blue outline in F). The stomach also appears slightly affected. (L) Three-dimensional reconstruction of histological sections illustrates the development of annular pancreas.|
|Fig. 1. Ventral pancreatic bud directs fusion of the dorsal and ventral pancreas. (A) Ventral halves of NF20 Elas-GFP transgenic embryos were recombined with dorsal halves of a wild-type embryo (VtgDwt) and grown to tadpole NF42. Only ventral pancreatic cells will be GFP+. The prospective dorsal and ventral pancreatic buds are illustrated. (B) Fluorescent image of an isolated NF42 whole gut, revealing GFP+ cells in the ventral pancreas, with punctate expression in the dorsal pancreas. (C) Isolated pancreas/liver tissue with no clear demarcation between the dorsal and ventral pancreas. Extensive GFP+ cells are found throughout the dorsal region of the fused pancreas. (D) Annular pancreas (AP) phenotype that developed in a VtgDwt recombinant. GFP+ cells are found in the annular pancreas. (E-G) Color-coded drawings of the images in B-D. Green, GFP+ transgenic pancreatic cells; blue, wild-type pancreatic cells; pink, liver; yellow, intestine. The region of the pancreas that lies behind the stomach is depicted using a lighter shade of green. (H) Schematic illustrating the recombination of dorsal wild-type and ventral transgenic halves (DtgVwt). Only dorsal pancreatic cells will be GFP+. (I) Fluorescent image of an isolated NF42 whole gut showing the fate of GFP+ dorsal pancreas cells. (J) Pancreas/liver tissue was isolated from the whole gut in order to better view the fate of GFP+ cells in the pancreas. There is a sharp demarcation at the dorsal-ventral border; gaps of GFPcells can be seen within the dorsal pancreas. (K) Annular pancreas that developed in a DtgVwt recombination. No GFP+ cells are found in the annular pancreas. (L-N) Color-coded drawings of the images in I-K.|
|Fig. 2. Ventral endoderm cells do not mix with the prepancreatic dorsal endoderm after the archenteron closes. (A) Fluorescent image of a VtgDwt chimeric embryo at NF32 showing GFP expression throughout the entire tadpole, but lacking in the dorsal region. (B) Isolated endoderm from NF35/36 VtgDwt chimeric tadpole showing GFP fluorescence throughout the ventral endoderm, but lacking in the dorsal-most region of the endoderm. (C) Double in situ hybridization for ptf1a (red) and GFP (purple) of endoderm shown in B. No gfp mRNA is detectable in the ptf1a expression domain. (D) Fluorescent image of a DtgVwt chimeric embryo at NF32 showing GFP expression only in the dorsal part of the tadpole. (E) Isolated endoderm from NF35/36 tadpole showing GFP fluorescence only in the dorsal layer of the endoderm. (F) In situ hybridization for ptf1a shown in G. The ptf1a expression domain is located in the dorsal layer of the endoderm. dp, dorsal pancreas.|
|Fig. 3. Microarray analysis of dorsal and ventral pancreatic buds. (A) Schematic illustrating the experimental plan. Individual dorsal and ventral pancreatic buds were isolated from NF38 tadpoles. (B) Diagram of isolated liver and pancreas tissue samples, illustrating the distinct dorsal and ventral regions of the early pancreas, shortly after fusion. Dark blue, dorsal pancreas; light blue, ventral pancreas; pink, liver. (C-F) Whole-mount in situ hybridization on isolated liver/pancreas tissue at NF40 of selected pancreatic genes. dp, dorsal pancreas. (G) RT-PCR analysis of selected pancreatic genes in dorsal and ventral pancreatic bud samples used for the microarray.|
|Fig. 4. Validation of microarray data. (A,B) Initial confirmation of the data by RT-PCR analysis of selected genes found to be enriched in either the dorsal or ventral bud fraction. (C-E) Dorsal-specific localization of brunol1, insm1 and secretogranin III in isolated liver/pancreas tissue at NF40, except for secretogranin III, which is at NF44. Expression in all three instances is punctate. (F-H) Ventral pancreas-specific expression of tm4sf3, pkc-alpha and agr2 at NF40/42. Expression is localized to a small region in the ventral pancreas, where the two ventral pancreatic buds fuse, and to where the bile duct emerges. Broken lines in C and F indicate the boundary of dorsal and ventral pancreas.|
|scg3 (secretogranin III ) gene expression in dissected Xenopus laevis gut, NF stage 41, as assayed by in situ hybridization. Lateral view: anterior left, dorsal up. Copyright Company Of Biologists Limited , 2009 Image extracted from XB-IMG-48831 and originally published in: Jarikji Z et al. (2009)|