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During blastula and gastrula stages of Xenopus development, cells become progressively and asynchronously committed to a particular germ layer. We have analysed the expression of genes normally expressed in ectoderm, mesoderm or endoderm in individual cells from early and late gastrula embryos, by both in situ hybridization and single-cell RT-PCR. We show that at early gastrula stages, individual cells in the same region may express markers of two or more germ layers, and ''rogue'' cells that express a marker outside its canonical domain are also observed at these stages. However, by the late gastrula stage, individual cells express markers that are more characteristic of their position in the embryo, and ''rogue'' cells are seen less frequently. These observations exemplify at the gene expression level the observation that cells of the early gastrula are less committed to one germ layer than are cells of the late gastrulaembryo. Ectodermal cells induced to form mesendoderm by the addition of Activin respond by activating expression of different mesodermal and endodermal markers in the same cell, recapitulating the response of marginal zone cells in the embryo.
Fig. 1. Rogue cells express Gsc, Xwnt8 and Sox17α in the Xenopus early gastrula. In situ hybridization using Gsc, Xwnt8 or Sox17α 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) Sox17α 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.
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, Sox17α,
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.
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.
Fig. 4. (A) Reproducibility of the single cell RTPCR
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 RTPCR,
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 RTPCR
reactions were analyzed together.
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).
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 Sox17α after 4 hours. (C) The gene
expression profile of individual cells at different times is shown in the presence and absence of Activin.