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A role for cytoplasmic determinants in mesoderm patterning: cell-autonomous activation of the goosecoid and Xwnt-8 genes along the dorsoventral axis of early Xenopus embryos.
Lemaire P
,
Gurdon JB
.
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Although an induction event is required for the formation of mesoderm in Xenopus embryos, it is not clear that this induction is wholly sufficient to give rise to a correctly patterned mesodermal layer. We have studied the expression of the two genes, goosecoid and Xwnt-8, in Xenopus gastrulae in which cell-cell communication, and therefore mesoderm induction, has been prevented by frequent cell dispersion. Although neither the early panmesodermal marker Xbra nor the muscle-specific alpha-actin gene were activated under these conditions, goosecoid and Xwnt-8 were activated in cells of dorsal and ventrolateral origin respectively, thus correctly reflecting their distribution during normal development. We also show that the spatial pattern of expression of these two genes along the animal-vegetal axis is similar in normal and in dissociated early gastrulae: goosecoid is mainly expressed in future mesoderm while the domain of expression of Xwnt-8 spans the mesoderm-endoderm boundary. These results show that, during the blastula and early gastrula stages, gene activation can be controlled cell-autonomously along both the animal-vegetal and dorsoventral embryo axes. This suggests that the inheritance of localised maternal cytoplasmic determinants is a key event for the patterning of mesoderm. We present a modified model of mesoderm formation in which the different mesoderm cell types are produced as a result of cooperation between induction-dependent and induction-independent immediate-early genes.
Fig. 1. Effect of cell dissociation and
reaggregation on the expression of Xbra, Xwnt-
8 and goosecoid. (A) Scheme of cell
dissociation; (B,C) analysis by RT-PCR of the
expression of Xbra, XWnt-8, goosecoid (gscd) and the FGF receptor (FGFR). The latter gene is
ubiquitously expressed and serves as a loading control. The samples analysed were eggs,
whole embryos (W), whole embryos dissociated between the stages 6 and 10.5 (WD), or whole
embryos dissociated between the stages 6 and 9 and subsequently reaggregated by the addition
of calcium and magnesium (WDR). For the analysis of gene expression, all embryos and
dissociated cells were lysed at the equivalent of stage 10.5.
Fig. 2. Expression of XWnt-8 and goosecoid in dissociated cells originating from the most dorsal or most ventral quarters of embryos.
(A) Experimental design. In the embryo diagrams, the dorsal side is to the left. The dorsal and ventral dye marks are shown in blue and red
respectively. (B) Histograms of the expression of Xwnt-8 or goosecoid shown as a percentage of the FGF receptor gene expression. The assay
used was RT-PCR. In this experiment, dissociation had an atypically strong effect on the level of expression of goosecoid in whole embryo
cells.
Fig. 3. Expression pattern of XWnt-8 and goosecoid in stage 10.5
dissociated cells descended from the vegetal, marginal and animal
parts of 32-cell embryos. (A) Fate map of the 32-cell embryo
indicating the percentage of the volume of the progeny of each of the
tiers A, B, C, and D that will become either mesoderm, ectoderm or
endoderm (data obtained from Dale and Slack, 1987b). AP, animal
pole; VP, vegetal pole. (B,C) RT-PCR analysis of the expression of
Xwnt-8, goosecoid (Gscd), Xbra or the FGF receptor (FGFR) in cells
originating from tier A, from pooled tiers B and C (B+C), or from
tier D of a 32-cell embryo. The analysis was performed on cells aged
to the equivalent of stage 10.5. W, whole stage 10.5 embryos; WD,
dissociated whole embryos aged to the equivalent of stage 10.5.
Fig. 4. Comparison of the expression patterns of XWnt-8 and Xbra
in normal early gastrulae. Non-radioactive probes for the genes
Xbra (A,D,G) or Xwnt-8 (B,E,H) were hybridised to alternate
sections of early Xenopus gastrulae. (C,F,I) Computer-generated
reconstructions of the nuclei staining for either Xbra (blue dots) or
Xwnt-8 (red dots) on the neighboring adjacent slides. In all panels,
the dorsal side is to the left and the ventral side to the right. Scale
bar, 200 um.
Fig. 5. Model of the early formation and patterning of Xenopus
mesoderm. See text for details. MIFs, mesoderm inducting factors.
