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First published online 12 September 2007
doi: 10.1242/dev.005280

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Induction of proepicardial marker gene expression by the liver bud

Yasuo Ishii, Jonathan D. Langberg, Romulo Hurtado^*, Sharrell Lee^* and Takashi Mikawa

University of California San Francisco, Cardiovascular Research Institute, Box 2711, Rock Hall Room 384D, 1550 4th Street, San Francisco, CA 94158-2324, USA.

View larger version (115K): [in this window] [in a new window]   Fig. 1. Close proximity between the PE and liver bud as shown molecularly by double in situ hybridization. (A-P) Chick embryos at stage 11 (M,N), stage 12 (I,J), stage 14 (A,B,E,F) and stage 16 (C,D,G,H,K,L,O,P) stained for expression of a PE marker gene (purple) [Wt1 (A-D), capsulin (E-H), Tbx18 (I-L) or Cfc1 (M-P)] in conjunction with for expression of a liver marker gene, Hex (turquoise). Right-side view (A-C,E-G,I-K,O) and ventral side view (M,N) of the whole-mount-stained embryos. B,F,J and N are high-magnification images of boxed regions in A,E,I and M, respectively. (D,H,L,P) Semi-sagittal sections of whole-mount-stained embryos at stage 16, showing that all PE marker genes are expressed in mesodermal tissue in direct contact with the Hex-positive liver bud. PE, proepicardium; LiB, liver bud; h, heart; im, intermediate mesoderm. Scale bars: 100 µm (A-C,E-G,I-K,M-O) and 20 µm (D,H,L,P).

View larger version (102K): [in this window] [in a new window]   Fig. 2. Expression of positive and negative PE markers. (A-E) Whole-mount in situ hybridization of chick embryos at stage 18 (A), stage 16 (B), stage 17 (C,E) and stage 15 (D) with probes for Wt1 (A), capsulin (B), Tbx18 (C), Cfc1 (D) and Pax2 (E). Arrows, proepicardium (PE); arrowheads, intermediate mesoderm. (F) A transverse frozen section through the PE of a stage 17 embryo. The section was immunostained with the anti-Wt1 antibody (green) and was counterstained with DAPI (blue). (G) A sister section of that shown in F stained with the anti-Pax2 antibody (green) and DAPI (blue). Notice that Wt1 but not Pax2 is detectable in the PE, whereas similar levels of staining are seen in the intermediate mesoderm.

View larger version (38K): [in this window] [in a new window]   Fig. 3. In vitro explant culture assay for PE marker induction. (A) Schematic of the assay. Chick lateral embryonic fragments (cLEF) were isolated from the fourth to seventh (4-7) somite levels of stage 10-11 embryos. Width of the somite(s) was used as a morphological reference for mediolateral levels of the fragment. The fragment was cultured alone, or co-cultured with the quail liver bud (qLiB) or lung bud (qLuB) in a hanging drop of M199. (B) Reverse transcriptase (RT)-PCR analysis of mRNA isolated from cultured explants. Weak signals for Wt1, capsulin and Pax2 are detectable in cLEF cultured alone. The level of signals for Wt1 and capsulin, but not Pax2, increased significantly when cLEF was co-cultured with the liver bud. In our RT-PCR condition, mRNAs of all these markers were undetectable in the liver bud cultured alone. (C) Quantification of PCR products of Wt1, capsulin and Pax2, showing enhancement of proepicardium (PE) marker expression in co-culture with the liver bud. Standard deviation bars are shown. (D) RT-PCR analysis using chick-specific primers for Wt1 (cWt1), Tbx18 (cTbx18) and Cfc1 (cCfc1). The liver bud has a strong capacity to upregulate PE marker gene expression in co-cultured cLEF. (E) Quantification of PCR products. We performed PCR changing the amount of the template cDNA to ensure linear amplification conditions. (F) Quantitative real-time (qRT)-PCR analysis of cWt1. Bars show the average of three independent PCR reactions.

View larger version (98K): [in this window] [in a new window]   Fig. 4. Ectopic expression of proepicardium (PE) marker genes in embryos implanted with the liver bud. (A) Schematic of the implantation procedure. Liver bud or lung bud was isolated from a quail donor and implanted ectopically in a host chick embryo. The host embryo was then cultured on an agar-albumen gel ventral side up. (B) A liver bud (arrow)-implanted embryo before whole-embryo culture. (C-J) In situ hybridization of liver bud-implanted embryos after 20 hours of whole-embryo culture. The embryos were stained for capsulin (C,D), Tbx18 (E,F), Pax2 (G,H) or Hex (I,J) transcripts. D,F,H and J are higher-magnification images of the boxed areas in C,E,G and I, respectively. Notice that ectopic expression of capsulin and Tbx18, but not of Pax2, is detectable at the site of implantation (arrows). The implant does not appear to affect endogenous expression for these markers in the intermediate mesoderm (im). (K-N) Lung bud-implanted embryos stained for capsulin (K,L) and Tbx18 (M,N). L and N are higher-magnification images of the boxed areas in K and M, respectively. No ectopic expression of these PE markers is detectable at implanted sites.

View larger version (117K): [in this window] [in a new window]   Fig. 5. Ectopic induction of Wt1 expression by the liver bud as shown by immunohistochemistry. (A) A chick embryo implanted with a quail liver bud (arrow) at stage 13; (B) after 20 hours of whole-embryo culture. (C-F) Transverse sections of the embryo shown in B at the level of the implant. The sections were stained with anti-Wt1 (C,D) or anti-Pax2 (E,F) antibody (green). The sections were also stained with a quail-specific QCPN antibody (red) and DAPI (blue; for counterstaining). D and F are high-magnification images of the boxed regions in C and E, respectively. Bona fide expression of Wt1 and Pax2 was detected in the intermediate mesoderm and dorsal mesothelium (asterisks). Wt1 (arrowheads in D), but not Pax2 (F), was detected ectopically in the ventrolateral lining of the body cavity. The signal was observed in the nuclei of the host-derived cells adjacent to the QCPN-positive donor tissue. (G-L) Embryos implanted with the liver at a more-posterior level, showing that ectopic Wt1 expression becomes detectable far form the intermediate mesoderm prior to the completion of the lateral body folding. im, intermediate mesoderm; nt, neural tube.

View larger version (71K): [in this window] [in a new window]   Fig. 6. Developmentally and spatially regulated inductive activity and competence. Embryos implanted with an inducer tissue (A,G,M,S) were cultured for 20 hours (B,H,N,T) and processed for immunohistochemistry. Transverse sections were cut and immunostained with anti-Wt1 (C,I,O,U) or Pax2 (E,K,Q,W) antibody (green), and with the QCPN antibody (red) and DAPI (blue). D,F,J,L,P,R,V,X are high-magnification images of the boxed regions in C,E,I,K,O,Q,U,W, respectively. Asterisks indicate bona fide expression of Wt1 or Pax2 in the intermediate mesoderm and dorsal mesothelium. nt, neural tube. (A-L) Inductive capacity of non-liver endodermal organs. (A-F) Embryo implanted with the lung bud. Ectopic induction of Wt1 expression is not detectable. (G-L) Embryo implanted with the stomach, showing a weak ectopic signal for Wt1 in a small number of cells in the host-derived tissue (arrowheads). No ectopic signal for Pax2 was detectable (E,F,K,L). (M-X) Developmetntally regulated inductive activity of the liver bud and of mesodermal competence to respond to this activity. (M-R) Young chick host (stage 11-) implanted with quail liver bud. Induction of Wt1 expression is not evident. (S-X) A stage 13 embryo implanted with an older (stage 21-22) liver bud. A low level of ectopic Wt1 expression in mesodermal tissue adjacent to the implant (arrowheads in V) was observed. No ectopic signal for Pax2 was detectable (Q,R,W,X).

View larger version (24K): [in this window] [in a new window]   Fig. 7. Summary of immunohistochemical analysis of implanted embryos. Each filled circle represents one implantation that induced ectopic Wt1 expression. Open circles represent implantations in which induction was not detectable. Different colors represent different implanted tissue. Gray filled circles represent implantations in which induced expression and endogenous proepicardium (PE) expression could not be distinguished because of their close proximity. Somite level and the distance between the midline and lateral edge of the dorsal aorta were used to determine anteroposterior and mediolateral levels of the implant. Quail-derived inducer tissues were implanted into stage 10--12- (left), stage 12-13- (middle) or stage 13-14 (right) chick host embryos at different sites. Induction of Wt1 expression in host tissue was examined by double immunohistochemistry using anti-Wt1 and QCPN antibodies.

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