QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 25 August 2004
doi: 10.1242/dev.01340

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wardle, F. C. Articles by Smith, J. C. Search for Related Content PubMed PubMed Citation Articles by Wardle, F. C. Articles by Smith, J. C. Social Bookmarking                         What's this?

Refinement of gene expression patterns in the early Xenopus embryo

Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK

View larger version (32K): [in a new window]   Fig. 6. Response of whole ectodermal explants to Activin. (A) Animal pole explants were dissected and incubated in the presence or absence of Activin protein for the indicated times. Explants were dissociated in CMFM at hourly intervals (0 hour time point is before addition of Activin) and single cells were picked for RT-PCR. Intact caps remaining at 4 hours were assayed by real-time RT-PCR. (B) Real-time RT-PCR of intact caps shows high levels of expression of XK70A and Xbra, lower levels of Mix.1, and almost no induction of Sox17 after 4 hours. (C) The gene expression profile of individual cells at different times is shown in the presence and absence of Activin.

View larger version (52K): [in a new window]   Fig. 2. (A) The single cell RT-PCR protocol. Ventral marginal zone (VMZ), dorsal marginal zone (DMZ), vegetal and animal pole explants are dissected and single cells dissociated in calcium/magnesium-free medium. Single cells are picked and subjected to RT-PCR. cDNA from each single cell is run on an agarose gel to check integrity and amplification, then Southern blotted or dot blotted, and probed with a specific marker. (B) Examples of dot blots. cDNA from each single cell is dotted onto a nylon filter in the format of a 96-well microtitre plate and probed for the markers indicated (ODC, Xbra, XK70A, Sox17, Xwnt8, Gsc). In this example, cells from stage 10 DMZ and VMZ explants are shown; column/row number corresponds to those shown for dorsal and ventral cells in Fig. 3.

View larger version (30K): [in a new window]   Fig. 4. (A) Reproducibility of the single cell RT-PCR protocol. Ventral marginal zone (VMZ) and dorsal marginal zone (DMZ) explants were dissected and single cells dissociated in calcium/magnesium-free medium. Single cells are picked and lysed, then the lysate was split into A and B samples. These samples were then subjected to RT-PCR in parallel. cDNA from each reaction was dot blotted onto a nylon filter and probed with a specific marker. (B) Examples of dot blots. cDNA from each single cell probed for the markers indicated (ODC, Mix.1, Gsc, Xbra). (C) Sensitivity of single cell RT-PCR protocol. Single cell lysates were spiked with 1, 10, 50, 100, 500, 1000 and 5000 transcripts of polyadenylated eGFP RNA and subjected to RT-PCR, dot blotted and probed for GFP. In two separate RT-PCR experiments, 10 transcripts of GFP were detected. No signal above background was detected in the no GFP RNA sample. (C) A dot blot where samples from separate RT-PCR reactions were analyzed together.

View larger version (77K): [in a new window]   Fig. 1. Rogue cells express Gsc, Xwnt8 and Sox17 in the Xenopus early gastrula. In situ hybridization using Gsc, Xwnt8 or Sox17 antisense probes on bisected embryos at early gastrula stage 10-10.5. (A-C) The majority of Gsc expression is seen in dorsal mesendoderm; however, some expressing cells (arrows) are seen in the ventral marginal zone (B') and the vegetal mass (A'-C'). (D-F) Xwnt8 expression is seen in the ventral marginal zone, although some expressing cells are seen in the dorsal marginal zone (D') and in the vegetal mass (E',F'). (G-I) Sox17 expression is strongest in the vegetal mass; however, discontinuous expression is also seen in the marginal zone (G'-I'). Dorsal is towards the left, animal towards the top. (A,D,E,G,H,I) The outside surface of embryo; (B,C,F) the cut surface of embryo. (A'-I'') Higher magnification of the cells indicated in A-I.

View larger version (54K): [in a new window]   Fig. 3. Gene expression profiles of single cells at the early gastrula stage; cells express a combination of markers. Animal, vegetal, DMZ and VMZ cells were isolated from stage 10 embryos and analyzed by dot blot or Southern blot (Fig. 2). Each block indicates the presence (coloured block) or absence (white block) of a marker gene. Each column represents the combination of marker genes seen in each cell. Those samples that did not show a signal for any marker are not included in the table. XK70A, an ectodermal gene, is represented in blue, mesodermal genes are represented in red, endodermal genes are represented in yellow, and Derrière, which is expressed in both mesoderm and endoderm, is represented in orange.

View larger version (51K): [in a new window]   Fig. 5. Gene expression profiles at the late gastrula stage are more uniform. (A) Animal, vegetal, DMZ and VMZ cells were isolated from stage 12 embryos and analyzed by RT-PCR and dot blot. (B) The gene expression profile of each cell is shown. In this figure cells with the same expression pattern have been grouped together rather than being shown in order of their position in a 96-well plate. (C) Gsc in situ hybridization on bisected embryos at stage 11.5 (parasagittal section, dorsal towards left) and stage 13 (sagittal section, anterior towards right, dorsal towards the top). At stage 11.5, Gsc is not detected in ectoderm by in situ hybridization, but by stage 13 a domain of Gsc expression can be seen in the neurectoderm (ec; arrow) overlying Gsc expression in the axial mesoderm (m).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter