Wacker SA et al. (2004), Timed interactions between the Hox expressing n...

XB-ART-3815

Dev Biol 2004 Apr 01;2681:207-19. doi: 10.1016/j.ydbio.2003.12.022.

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Timed interactions between the Hox expressing non-organiser mesoderm and the Spemann organiser generate positional information during vertebrate gastrulation.

Wacker SA , Jansen HJ , McNulty CL , Houtzager E , Durston AJ .

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We report a novel developmental mechanism. Anterior-posterior positional information for the vertebrate trunk is generated by sequential interactions between a timer in the early non-organiser mesoderm and the organiser. The timer is characterised by temporally colinear activation of a series of Hox genes in the early ventral and lateral mesoderm (i.e., the non-organiser mesoderm) of the Xenopus gastrula. This early Hox gene expression is transient, unless it is stabilised by signals from the Spemann organiser. The non-organiser mesoderm and the Spemann organiser undergo timed interactions during gastrulation which lead to the formation of an anterior-posterior axis and stable Hox gene expression. When separated from each other, neither non-organiser mesoderm nor the Spemann organiser is able to induce anterior-posterior pattern formation of the trunk. We present a model describing that convergence and extension continually bring new cells from the non-organiser mesoderm within the range of organiser signals and thereby create patterned axial structures. In doing so, the age of the non-organiser mesoderm, but not the age of the organiser, defines positional values along the anterior-posterior axis. We postulate that the temporal information from the non-organiser mesoderm is linked to mesodermal Hox expression.

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Species referenced: Xenopus
Genes referenced: egr2 en2 hoxa7 hoxb4 hoxb9 hoxc6 hoxc9-like hoxd1 hoxd13 nog nrp1 tbx2 tbxt

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	Fig. 1. Spatial and temporal Hox expression during gastrulation. Results for Hoxd-1, Hoxb-4, Hoxc-6, Hoxa-7 and Hoxb-9 are shown, Hoxa-1, Hoxb-1 and Hoxb-7 also fit this sequence (not shown), Hoxd-13 expression did not begin before the end of gastrulation (stage 15, not shown). (A) WISH for five Hox genes at five different stages. Vegetal views, dorsal up. (B) Diagram showing the onset of the temporally colinear expression of five different Hox genes as analysed with WISH (red) and PCR (blue). (C) Localisation of Hoxd-1 expression in mesoderm and ectoderm. Embryos cut into halves across the dorsolateral blastopore lip at stages 10.5 and 11. One half analysed with Xbra, the other with Hoxd-1. The Xbra expression domain is outlined. The initial Hoxd-1 expression is located within the mesoderm. At stage 11, it is expanded into the presumptive neurectoderm (arrowheads).
	Fig. 2. Hox expression in ventralised and dorsalised embryos. (A) Hoxc-6 in embryos ventralised with UV. WISH (vegetal views of stages 10.5 to 12.5, lateral view of stage 26). Similar results were obtained for Hoxd-1, Hoxb-4, Hoxa-7 and Hoxb-9 (not shown). (B) Diagram showing the onset of Hox expression (detected with WISH) in ventralised (dark blue) and control embryos (light blue). In ventralised embryos, the temporally colinear sequence is still present. (C) Mesodermal Hox expression (WISH) in ventralised embryos. Embryos cut through dorsolateral blastopore lips in controls and the corresponding region in ventralised embryos. Hoxd-1 at stage 11, Hoxc-6 at stage 12, Hoxa-7 at stage 12.5. The white line indicates Brachet’s cleft, separating involuted mesoderm from overlying ectoderm. Neurectodermal Hox staining in control embryos (C, arrowheads) is missing in ventralised embryos (C’). (D) Hox expression is absent in embryos that were dorsalised with LiCl. Dorsalised embryos (LiCl) and controls (con) at stage 12.5 (vegetal views) and stage 26 (lateral views). Results of a WISH for Hoxd-1, Hoxc-6 and Hoxb-9. Analysis of Hoxb-4 and Hoxa-7 showed similar results (not shown). Arrowheads indicate the anterior Hox expression boundary.
	Fig. 3. Recombinations of Spemann organiser (SO) and ventralised embryos (containing only non-organiser mesoderm) as indicated in the schematic drawing (A). (B –F) Stage 10 to 10+ organiser tissue was implanted into the marginal zone of stage 10 ventralised embryo. Analysis at stage 27 (lateral views). A nontreated control embryo (con), a ventralised embryo without graft (UV) and ventralised embryos implanted with organiser mesoderm (UV + SO). Probe combinations are indicated. Arrowheads show the distance between the most posterior Krox-20 expression and the anterior Hox expression boundary. The normal spatially colinear Hox sequence is restored by organiser transplantation. (G–N) Organiser tissue was explanted from dorsal blastopore lips of stage 10 to 10+ and stage 11.5, respectively, and implanted into the marginal zone of stage 10+ ventralised embryos. Analysis at stage 27 (lateral views). Shown are nontreated controls (con), ventralised embryos without grafts (UV) and recombinations with early organiser (10 + UV + 10 + SO) and late organiser (10 + UV + 11.5 SO), respectively. Probe combinations are indicated. Arrowheads show the distance between the most posterior Krox-20 expression and the anterior Hox expression boundary.
	Fig. 4. Timed interactions between the Spemann organiser and the non-organiser mesoderm (NOM). (A) Ageing the non-organiser mesoderm (isolated in ventralised embryos). A ventralised embryo with no implant (UV), an untreated control embryo (con), and recombinations of organiser mesoderm from stage 10 (0h SO) with ventralised embryos of different ages after the beginning of gastrulation (0h, 2h, 4h, 6h NOM). Embryos are positioned with their head up and dorsal to the right. They were analysed with WISH using axial markers, including En-2 (midbrain –hindbrain border), Krox-20 (hindbrain), Hoxb-4 (posterior hindbrain), Hoxc-6 and Hoxa-7 (anterior spinal cord), Hoxd-13 (posterior spinal cord). Expression of Krox-20 (arrow heads) and Hoxd-13 illustrates the results. Pictograms indicate restored part of axis (based on conclusion from all markers). (B) Ageing the Spemann organiser. A ventralised embryo without implant (UV), an untreated control (con), and recombinations of stage 10 ventralised embryos (0h NOM) with organiser tissue (SO) aged for 0h, 2h, 4h, 6h after beginning of gastrulation. Embryos orientated and WISH analysed as in (A). Krox-20 expression (arrowheads) and Hoxd-13 illustrate the results. Pictograms indicate restored part of axis. The age of the organiser implant does not affect the restored axial values. (C) Timed restoration of organiser functions by Noggin protein (nog) injection. Ventralised embryos were injected with Noggin protein into the blastocoel (schematic drawing) at different blastula and gastrula stages. Embryos were analysed as above. Left panel stained for En-2/Krox-20/Hoxc-6/Xbra, right panel for Krox-20/Hoxd-13. Embryos are orientated as in (A), arrows point to Krox-20 expression. Top, non-injected ventralised embryos (UV). Rows 2– 5 show ventralised embryos injected with Noggin at the indicated stages. Bottom, control embryos (con). Early-treated embryos restore head (grey colour in the corresponding pictograms) and anterior trunk (Krox-20 expression, blue colors in pictograms). Later-treated embryos show progressively less head (grey) and more trunk (anterior trunk marked by Krox-20 and blue colors in pictograms, posterior trunk marked by Hox genes and Xbra, yellow and red colors in pictograms). Very late on, there is an extensive zone of Hoxd-13 expression (posterior trunk) and anterior trunk markers (e.g., Krox-20) have reached the anterior end of the embryo.
	Fig. 5. Neurectodermal Hox expression requires signals from organiser mesoderm and non-organiser mesoderm. (A) Wrap assay. Spemann organiser tissue (SO) and/or non-organiser mesoderm (NOM) are wrapped in two (ectodermal) animal caps (AC). (B) Wrap assays (fixed around the end of gastrulation) were dissected and analysed for ectodermal Hoxd-1 expression using WISH. Tissue localisation is indicated in the corresponding schematic drawings. Hoxd-1 expression: blue stipples. Only combined Spemann organiser (SO) and non-organiser mesodermal tissue (NOM) induce Hoxd-1 expression (arrowheads) in ectodermal animal caps (AC). (C–D) Ectodermal lineage tracing inWraps containing non-organiser and organiser mesoderm. A Wrap after in situ hybridisation for Hoxd-1 (arrowheads in C) and the corresponding fluorescence staining. In the magnified sectors, the arrowheads indicate that the tissue borders in the Wraps correspond to the borders between mesodermal implant and fluorescence labelled ectoderm. The main portions of Hoxd-1 staining are ectodermal.
	Fig. 6. The time space translator model. (A) False colour representation of expression of three Hox genes during gastrulation. WISH on sibling embryos for Hoxd-1 (purple), Hoxc-6 (green), Hoxb-9 (red). Digital images were analysed and selected areas labelled with respective false colour and combined in one image. Six gastrula stages (10.5, 11, 11.5, 12, 12.5 and 13) are shown in a lateral view, anterior up and dorsal to the right. Anterior levels of the Hox expression at the end of gastrulation are arrowed. (B) The time space translator model. Expression of new Hox genes (different colours) is initiated in non-organiser mesoderm (NOM) at different times. Non-organiser mesodermal tissue moves toward the Spemann organiser by convergence and then extends anteriorly (arrow). When mesoderm adjacent to the Spemann organiser involutes (lM), the current Hox code is transferred to overlying neurectoderm (NE). While the early Hox sequence in the non-organiser mesoderm (solid outlined black box) is running, new cells from this region are continuously moved into the range of Spemann organiser (dashed black box) and their Hox code is then stabilised by an organiser signal. Thus, the temporal Hox sequence is converted into a spatial AP pattern by continuous morphogenetic movement and stabilisation of timed information by the organiser in both involuted mesoderm (IM) and overlying neurectoderm (NE). (C) Dorsal views. In non-organiser mesodermal cells, the Hox sequence is running (solid black outline). From this domain, cells are continuously moved into influence of Spemann organiser (dashed black box) by convergence and extension (arrows). The AP pattern arises by adding new stabilised segments expressing a different subset of Hox genes posteriorly. A, anterior; P, posterior; V, ventral; D, dorsal; L, left; R right. (D) Schematic diagrams depicting locations of Spemann organiser, blastopore and initial Hox expression domain in Xenopus and orthologous structures in the zebrafish (Alexandre et al., 1996), the chick (Gaunt and Strachan, 1996) and the mouse (Deschamps et al., 1999) at the beginning of gastrulation. Zebrafish and Xenopus are shown in vegetal views, chick and mouse are shown in dorsal views.