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The formation of the vertebrate body axis during gastrulation strongly depends on a dorsal signaling centre, the Spemann organizer as it is called in amphibians. This organizer affects embryonic development by self-differentiation, regulation of morphogenesis and secretion of inducing signals. Whereas many molecular signals and mechanisms of the organizer have been clarified, its function in anterior-posterior pattern formation remains unclear. We dissected the organizer functions by generally blocking organizer formation and then restoring a single function. In experiments using a dominant inhibitory BMP receptor construct (tBr) we find evidence that neural activation by antagonism of the BMP pathway is the organizer function that enables the establishment of a detailed anterior-posterior pattern along the trunk. Conversely, the exclusive inhibition of neural activation by expressing a constitutive active BMP receptor (hAlk-6) in the ectoderm prohibits the establishment of an anterior-posterior pattern, even though the organizer itself is still intact. Thus, apart from the formerly described separation into a head and a trunk/tailorganizer, the organizer does not deliver positional information for anterior-posterior patterning. Rather, by inducing neurectoderm, it makes ectodermal cells competent to receive patterning signals from the non-organizermesoderm and thereby enable the formation of a complete and stable AP pattern along the trunk.
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17703924
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Fig. 1. The Spemann organizer induces a rudimentary AP pattern in wrap assays. (A) In the wrap assay small mesodermal explants (mes, e.g. organizer or non-organizermesoderm) are wrapped into ectodermal animal caps (ect) to analyze inductive events. (B) A wrap in topview and lateral view. For the analysis Wraps are bisected after in situ hybridization across the implants (dashed line). (C) Both halves of a bisected Wrap. Vital staining of two mesodermal implants (red; organizermesoderm (SO), green; non-organizermesoderm (NOM)) in unlabeled ectoderm show that the tissues do not intermingle. (D–F) The general neural markers Nrp-1, Sox-2, and Sox-3 are activated in Wraps containing exclusively organizer implants. (G) The forebrain marker Xotx-2 is induced in the ectoderm of wraps by an organizer implant. (H) En-2, a marker for the midbrain–hindbrain boundary, is weakly induced (arrowheads) by the organizer. I Krox-20, which is normally expressed in the rhombomeres 3 and 5 of the hindbrain, is not activated by the organizer. (J–L) The Hox genes Hoxd-1, Hoxc-6, and Hoxb-9, which label different positions along the AP axis are not induced in Wraps containing exclusively organizermesoderm.
Fig. 2. UV treatment results in embryos without an organizer. Embryos were injected with EosFP. The EosFP in a small region of the marginal zone was converted at the beginning of gastrulation. (A) An untreated control embryo (CON) with a conversion in the organizer region. (B) An UV-treated embryo (UV) with a corresponding conversion of the marginal zone. (C) Shape of the conversion at late gastrulation in an untreated control embryo. (D) Shape of the conversion at late gastrulation in an UV-treated embryo. (E) Bisection of the control embryo shown in (A). (F) Bisection of the UV-treated embryo shown in (B). (G) Bisection of the control embryo shown in (C). Arrowheads indicate Brachet’s cleft between not involuted and involuted tissue. (H) Bisection of the control embryo shown in (D). Arrowheads indicate Brachet’s cleft. The arrowheads in (A–D) indicate the plane of bisection. Comparison of gene expression in control embryos and UV-treated embryos. (I, J) The expression of the organizer gene Goosecoid (Gsc) at early gastrulation in an untreated control (I) and in an UV-treated embryo (J). (K, L) The expression of the organizer gene Chordin (Chd) at mid to late gastrulation in an untreated control (K) and in an UV-treated embryo (L). (M) The mesodermal marker Brachyury (Xbra) is expressed around the blastopore and in the forming notochord. (N) In UV-treated embryos the Xbra expression around the blastopore is found, the notochordal expression is absent. (O) A cross section of an untreated control embryo labeled for lateral plate mesoderm with FoxF1 (stage 26). (P) A cross section of an UV-treated embryo labeled for the lateral plate mesoderm with FoxF1 (stage 26).
Fig. 3. Restoration of neural activation by injection of tBr and FGF-4 in predominantly ectodermal precursors of UV-treated embryos results in AP patterning of embryos without Spemann organizer. The first column shows expression of different markers in untreated control embryos (CON). The second column shows expression of these markers in UV-treated embryos (UV). The third column shows expression of these markers in UV-treated embryos, which were animally injected with mRNAs for tBr and FGF-4 (UV + tBr/FGF-4). (A–C) Vegetal view of early gastrula embryos stained for the organizer gene Goosecoid (Gsc). In UV-treated embryos gsc is not expressed. Its expression is not restored by the animal injection of tBr and FGF-4, indicating the remaining absence of an organizer. (D–F) Vegetal view of stage 11.5 embryos stained for Chordin (Chd), which normally is expressed in the organizer and the overlying neural floor plate. In UV-treated embryos it is absent and not restored after tBr and FGF-4 mRNA injection into the animal pole, indicating the remaining absence of the organizer. (G–I) Expression of the mesodermal marker Brachyury (Xbra) at stage 12.5 in the marginal zone around the blastopore and in the prospective notochord (arrowhead). The notochordal expression is absent in UV-treated embryos and not restored after injection of tBr and FGF-4 mRNAs, again indicating the absence of the organizer. The non-organizer expression around the blastopore remains. (J–L) Sox-2 expression demarcates the neural plate in control embryos, and indicates the absence of neurectoderm in UV-treated embryos (stage 15). After injection of tBr and FGF-4 into the animal region of UV-treated embryos, almost all ectoderm shows Sox-2 expression. (M–O) Hoxd-1, which is expressed up to the level of the posterior portion of hindbrain, is absent in UV-treated embryos, but is mildly restored after injection of tBr and FGF-4 mRNAs in ectodermal precursors of UV-treated embryos (stage 15). (P–R) Hoxc-6, which is expressed along the spinal cord, is depleted in UV-treated embryos. Expression is restored after injection of tBr and FGF-4 mRNA in the animal blastomeres of UV-treated embryos (stage 15). (S–U) Hoxa-7 expression along the spinal cord of control embryos (stage 18) is depleted in UV-treated embryos, but is restored after injection of tBr and FGF-4 mRNA in animal blastomeres of UV-treated embryos. (V–X) Hoxb-9 expression along the posterior spinal cord of control embryos (stage 18) is depleted in UV-treated embryos, but is restored after injection of tBr and FGF-4 mRNA in animal blastomeres of UV-treated embryos.
Fig. 4. The Spemann organizer function for AP patterning in wrap assays can be replaced by neural activation. (A–C) Wraps containing both, organizer and non-organizermesoderm (AC + SO + NOM) express Hoxd-1 (A), Hoxd-4 (B) and Hoxb-9 (C) in the ectoderm. (D–F) Wraps containing exclusively non-organizermesoderm (AC + NOM) do not express Hoxd-1, Hoxd-4, or Hoxb-9. The same observation was made for Wraps containing exclusively organizermesoderm (not shown). (G–I) Neural activation by tBr and FGF-4 mRNA in the ectoderm of wraps without mesodermal implants (AC(tBr/FGF)) does not result in expression of Hoxd-1, Hoxd-4, or Hoxb-9. (J–L) tBr and FGF-4 mRNA injection to neurally activate ectoderm of Wraps containing non-organizermesoderm (AC(tBr/FGF) + NOM) replaces the organizer function for induction of the expression of Hoxd-1, Hoxd-4, or Hoxb-9 (arrowheads).
Fig. 5. Ectopic areas of neural activation after injection of tBr and FGF-4 in a ventral animal blastomere at 32-cell stage show AP patterning gene expression independent of the organizer. (A, G) Lineage tracing with GFP (arrowheads) after coinjection of the mRNAs of tBr, FGF-4, and EGFP. (A) Ventrolateral view at stage 18. The head is to the left. (B) The embryo from (A) after in situ hybridization for the neural marker Sox-2 shows ectopic ventrolateral Sox-2 expression (arrowhead). (C) Ectopic expression of tBr and FGF-4 results in an ectopic Hoxd-1 domain at stage 18 (arrowhead). (D) Ectopic expression of tBr and FGF-4 results in an ectopic Hoxd-4 domain at stage 18 (arrowhead). (E) Ectopic expression of tBr and FGF-4 results in an ectopic Hoxc-6 domain at stage 18 (arrowhead). (F) Ectopic expression of tBr and FGF-4 results in an ectopic Hoxb-9 domain at stage 18 (arrowhead). (G) A lateral view at stage 28. The head is up and ventral to the left. The arrowhead indicates the GFP domain. (H) Lateral view of a normal embryo at stage 30 showing Hoxd-4 expression. (I) Lateral view of an injected embryo at stage 30 showing an ectopic ventral patch of Hoxd-4 expression (arrowhead).
Fig. 6. Inhibition of neural activation in presence of an organizer strongly affects AP pattern formation. The mRNA of the constitutively active BMP receptor hAlk-6 was injected into predominantly ectodermal precursors of the left side (arrowheads) to prevent neural activation on one side of the embryos. (A, B) Presence of the early Gsc expression in a non-injected control embryo (CON) and in a hAlk-6 injected embryo (Alk-6) demonstrates that the formation of an organizer is not disturbed by the injection of hAlk-6. (C, D) Presence of Chd expression in a non-injected control embryo and in a hAlk-6 injected embryo at the end of gastrulation demonstrates the remaining presence of the organizer during gastrulation. (E, F) Expression of the mesodermal marker Xbra in the organizertissue of the presumptive notochord again shows the presence of the organizer both, in a non-injected control and in a hAlk-6 injected embryo. (G, H) The posterior marker Xwnt-8 remains in its domain on the injected side, indicating that hAlk-6 expression does not prevent formation of posterior tissues. This is in accordance with the minorly affected posteriorXbra expression around the blastopore (E, F). (I, J) The neural marker Sox-2 is expressed in the whole neural plate of control embryos, but it is drastically reduced after hAlk-6 injection, indicating the inhibition of neural activation on the injected side. (G, H) Expression of Hoxd-1. (I, J) Expression of Hoxc-6. (K, L) Expression of Hoxb-9. The expression of all three Hox genes is reduced on the left side, where neural activation is inhibited by hAlk-6 injection.
Fig. 7. Inhibition of neural activation by ectodermal expression of the constitutive active BMP receptor hAlk-6 in wrap assays affects the ectodermal AP patterning. (A) Expression of Hoxd-1 (arrowheads) in wraps containing both, organizer and non-organizermesoderm surrounded by non-injected ectoderm (AC + SO + NOM). (B) Inhibition of neural activation by injection of hAlk-6 in the ectoderm of such wraps (AC(Alk) + SO + NOM) results in the repression of Hoxd-1 expression. (C) Expression of Hoxc-6 (arrowheads) in wraps containing organizer and non-organizermesoderm surrounded by non-injected ectoderm. (D) Inhibition of neural activation in the ectoderm of such wraps results in repression of Hoxc-6 expression. (E) Expression of Hoxb-9 (arrowheads) in wraps containing organizer and non-organizermesoderm surrounded by non-injected ectoderm. (F) Inhibition of neural activation in the ectoderm of such wraps results in the repression of Hoxb-9 expression.
Supplementary Figure. Marker analysis of UV-treated embryos. A The marker Sox-2 demarkates the neural plate in untreated control embryos (CON) at stage 17 B Sox-2 is absent in UV treated embryos (UV). C The marker Xsna demarcates the neural crest of non-treated control embryos (arrowheads). D In UV treated embryos Xsna is not expressed indicating the absence of neural crest. E The epidermal marker XK81A1 is expressed in non-neural ectoderm of non-treated control embryos. F In UV treated embryos XK81A1 is expressed in all ectodermal cells indicating the absence of the neural plate. G Cross sections show that brevican (Xbcan) is expressed in the notochord (arrowhead) of non-treated control embryos at stage 26, which originates from the organizer. H In cross sections of UV treated embryos of an identical stage no Xbcan is found. I The somitic mesoderm marker MyoD is expressed in the forming somites on both sides of the dorsal midline in non-treated control embryos at stage 12.5. J In UV treated embryos the MyoD expression is reduced to a small domain around the blastopore. K Cross sections of non-treated control embryos at stage 26 show Xwnt-8 expression (arrowheads) in the intermediate mesoderm. L In cross sections of UV treated embryos Xwnt-8 (arrowheads) is expressed as a stripe around the blastopore indicating that there is still intermediate mesoderm. M, N Xotx-2 expression in the headmesoderm and the anterior portion of the neural plate is downregulated in UV treated embryos indicating the absence of organizer derived headmesoderm and of neural activation. O In non-treated control embryos (stage 20, anterior view) En-2 expression demarcates the mid-/hindbrain boundary. P In UV treated embryos the En-2 expression is absent. Q Hoxd-4 is expressed along the spinal cord in non-treated control embryos (stage 20, anterior view). R In UV treated embryos Hoxd-4 expression was not detected.
