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First published online 7 January 2004
doi: 10.1242/dev.00963

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Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in Arabidopsis thaliana

Achim Haecker^*, Rita Groß-Hardt, Bernd Geiges, Ananda Sarkar, Holger Breuninger, Marita Herrmann and Thomas Laux

Institute of Biology III, University of Freiburg, Schänzlestraße 1, 79104 Freiburg, Germany

View larger version (49K): [in a new window]   Fig. 1. Comparison of the WUS protein family. (A) Alignment of the homeodomain sequences. Asterisks indicate residues that are highly conserved in homeodomains (Gehring et al., 1990). (B) Alignment of the WUS box that is located downstream of the homeodomain. No WUS box was found in WOX13 (dashed line). (C) Alignment of the C-terminal region in a subset of the WUS family. In A-C identical residues are in black boxes, conservative changes in gray. (D) Dendrogram based on the sequence of the homeodomains. Bootstrap values are given (1000 rounds). AmWUS, PhWUS, LeWUS, OzWUS, putative orthologs of Antirrhinum, Petunia, tomato, and rice, respectively. See text for details and references.

View larger version (16K): [in a new window]   Fig. 2. Arabidopsis ovule organization and early embryo development. The green rectangle indicates the part of the ovule shown in Fig. 3. See text for details. a, apical daughter of the zygote; ad, apical embryo domain; b, basal daughter of the zygote; cd, central embryo domain; cc, central cell (blue); c, cotyledons; ec, egg cell (red); hy, hypophysis; in, integuments; pd, protoderm; ram, root apical meristem; sc, synergid cells (yellow); sam, shoot apical meristem. The demarcation line between the apical and central domain of the early embryo is indicated (open arrowheads).

View larger version (92K): [in a new window]   Fig. 3. WOX2 and WOX8 mRNA expression patterns (brown) in the female gametophyte. In situ hybridization of unfertilized ovules. Sections showing the mature embryo sac as outlined in Fig. 2. (A) Hybridization with a WOX2 antisense probe. WOX2 mRNA is detected in the egg cell and the central cell, but not in the synergids. (B) Hybridization with a WOX8 antisense probe. WOX8 mRNA is also detected in the egg cell and the central cell, but not in the synergids. (C) Hybridization with a WOX8 sense probe. No staining was detected in any of the cells of the embryo sac. cc, central cell; ecn, egg cell nucleus; sc, synergid cells. Scale bars: 10 µm.

View larger version (123K): [in a new window]   Fig. 4. mRNA expression patterns during early embryogenesis. (A-U) Longitudinal median sections of developing wild-type seeds. Bound probe is stained brown. The clonal boundary resulting from the division of the zygote is marked (black arrowheads). The demarcation line between the apical and central domain of the early embryo is indicated (open arrowheads). The white asterisks indicate brown color of the seed coat independent of the staining reaction. (A-G) In situ hybridization with a WOX2 antisense probe. (A) WOX2 mRNA is detected in the zygote. (B) After the first zygotic division, WOX2 expression is restricted to the apical daughter cell, and absent from the basal one. (C) WOX2 mRNA is expressed in all cells of the 4-cell embryo proper (unspecified arrow), but not in the suspensor. (D) WOX2 mRNA is expressed in the apical domain of the 8-cell embryo, but not in the central domain, the suspensor or the hypophysis. (E-F) At the 16-cell stage (E) and in early globular embryos (F), WOX2 mRNA remains restricted to the apical embryo domain. (G) In heart stage embryos, WOX2 expression is only weakly detected in epidermal cells of the central domain (arrow). (H-N) Hybridization with a WOX9 antisense probe. (H) No expression is detectable in the zygote. (I) WOX9 mRNA is detected only in the basal daughter cell of the zygote. (J) At the 4-cell stage, WOX9 expression is restricted to the hypophysis. (K) In 8-cell embryos, WOX9 mRNA is expressed in the central embryo domain and in the hypophysis. (L) In 16-cell embryos, WOX9 mRNA expression is restricted to epidermal cells of the central domain and to the hypophysis (arrows). (M) In mid globular embryos, WOX9 expression is detected in epidermal cells of the central domain, but not in the hypophysis (arrow). (N) In heart stage embryos, WOX9 mRNA is only detected in a ring of epidermal cells (arrow). (O-U) Hybridization with a WOX8 antisense probe. (O) WOX8 mRNA is detected in the zygote. (P) After the first division of the zygote WOX8 mRNA is exclusively found in the basal daughter cell but not in the apical one. (Q,R) WOX8 expression marks all suspensor cells of the 4- and 8-cell embryo, but is not expressed in the embryo proper (unspecified arrow in Q). (S) At the 16-cell stage, WOX8 mRNA is detected in all suspensor cells, including the hypophysis. (T) In mid globular embryos, continuous strong WOX8 expression is detected in the suspensor, but there is no expression in the derivatives of the hypophysis. (U) In heart stage embryos, WOX8 is expressed in the suspensor and surrounding endosperm cells. a, apical daughter cell of the zygote; ad, apical domain of the early embryo; b, basal daughter cell of the zygote; cd, central domain of the early embryo; en, endosperm; hy, hypophysis; su, suspensor; z, zygote. Scale bars: 10 µm (A-T): 20 µm (U).

View larger version (175K): [in a new window]   Fig. 5. WOX9 mRNA expression in mutant embryos. (A-F) In situ hybridizations with a WOX9 antisense probe (brown staining). Longitudinal sections of developing seeds. The demarcation line between the apical and central domain of the early embryo is indicated (open arrow heads). White asterisks indicate brown color of the seed coat independent of the staining reaction. (A,B) bdl embryos. WOX9 mRNA is detected in the hypophysis, but not in the central domain of double octant embryos corresponding to the 8-cell stage wild type (A, compare to Fig. 4K) and early heart stage (B, compare to Fig. 4M,N) of wild-type siblings. (C) mp embryo. WOX9 mRNA is detected in the hypophysis, but not in the central domain of embryos corresponding to the heart stage of wild-type siblings (compare to Fig. 4M,N). (D) WOX9 expression in a gn embryo; the stage shown corresponds to the heart stage of wild-type siblings (compare to Fig. 4N). WOX9 mRNA is detected in several epidermal cells throughout the embryo (arrow), but not in the hypophysis. (E,F) WOX9 expression in wild-type embryos, with abnormal cell division patterns. (E) A double octant embryo resembling that of bdl. WOX9 mRNA is detected in the central domain. (F) An abnormal embryo, corresponding to the late globular stage. Expression of WOX9 is roughly similar to that of normally developed wild-type embryos (compare to Fig. 4M). ad, apical embryo domain; cd1 and cd2, doubled central domain of double octant embryos; hy, hypophysis; su, suspensor. Scale bars: 20 µm.

View larger version (105K): [in a new window]   Fig. 6. WOX5, PRS/WOX3 and WOX1 mRNA localization (brown staining) during embryonic development. (A-L) Longitudinal median sections of developing wild-type seeds are shown, if not indicated otherwise. White asterisks indicate brown color of the seed coat independent of the staining reaction. (A-F) In situ hybridization with a WOX5 antisense probe. (A) No expression is detected in 16-cell embryos. (B) In early globular embryos, expression is detected exclusively in the hypophysis. (C) After the division of the hypophyseal cell, WOX5 expression becomes restricted to the upper daughter, the lens shaped cell (lsc), and is not detectable in the lower daughter, the columella precursor (cp). (D,E) In heart stage (D) and bent cotyledon stage (E) embryos, WOX5 mRNA is detected in the derivatives of the lens shaped cell, the developing quiescent center of the embryonic root (arrows). (F) Cross section through cotyledon primordia of a torpedo stage embryo (plane of section shown in J). WOX5 expression is detected in patches at about the position where lateral veins will form (arrow). (G-I) In situ hybridization with a PRS/WOX3 antisense probe. (G,H) Early heart stage embryos. (G) Expression is detected in a subset of cotyledonary cells (arrow). (H) Section through a cotyledon as indicated by a line in G). PRS/WOX3 mRNA is detected in two lateral stripes. (I) A torpedo stage embryo. PRS/WOX3 mRNA is restricted to a single cell at the tip of the cotyledon (arrow). (J-L) In situ hybridization with a WOX1 antisense probe. (J) A heart stage embryo. WOX1 mRNA is detected in the provascular tissue of the cotyledons, but not in the vasculature of the hypocotyl (arrow). (K) Cross section through a torpedo stage cotyledon as indicated by the line in J. WOX1 expression is detected in a stripe across the cotyledon. (L) Bent cotyledon stage. WOX1 signal is weaker, but still detectable in the provascular tissue of the cotyledons (arrow). c, cotyledon, cp, columella primordium; hy, hypophysis; lsc, lens shaped cell; su, suspensor. Scale bars: 20 µm.

View larger version (89K): [in a new window]   Fig. 7. wox2 embryos show specific defects in early development. (A) The gray bar indicates the WOX2 genomic DNA fragment used in rescue experiments. Above it, exon 1 and exon 2 have been enlarged to illustrate the positions of insertions within WOX2. (B-D) Wild-type embryos display a regular cell division pattern. (B) An 8-cell embryo. (C) An early globular stage embryo. (D) A mid-globular stage embryo. (E-G) By contrast, about 30-50% of wox2-1 embryos display defective apical development. (E) A wox2-1 embryo at the same stage as the one shown in B, where the left cell failed to divide (arrow). (F) A wox2-1 embryo at the same stage as the ones shown in C, displaying aberrant oblique cell divisions in the apical embryo domain (arrow). (G) A wox2-1 mid globular stage embryo. A protoderm has formed despite aberrant oblique cell divisions in the apical embryo domain (arrow). (H-J) About 15-30% of wox2-2 embryos have similar defects to those of wox2-1 embryos. (H) 2-cell embryo, showing a horizontal division (arrow) instead of a typical vertical one. (I) Early globular stage embryo, displaying an oblique cell division (arrow). (J) Mid stage embryo, displaying a misplaced tangential cell division (arrow) (B-G) DIC microscopy. Scale bars: 20 µm.

View larger version (10K): [in a new window]   Fig. 8. WOX gene expression dynamics during early embryo patterning. First, the egg cell and the zygote express WOX2 and WOX8. A hypothetical polar distribution of the respective RNAs, or factors regulating their expression, is shown. After division of the zygote, WOX2 expression is restricted to the apical daughter cell, which will give rise to the embryo proper, whereas WOX8 expression, together with WOX9, is restricted to the basal daughter cell, which will form the suspensor and the hypophysis. At the 8-cell stage, the basic domains of the embryo can be distinguished by different WOX gene expression profiles: (1) the apical domain that will give rise to most of the shoot expresses only WOX2, (2) the central domain that will form the hypocotyl and part of the root expresses WOX9 (and briefly low levels of WOX2 not illustrated here), (3) the hypophysis from which part of the root meristem will derive expresses WOX8 and WOX9, and (4) the suspensor expresses only WOX8. a, apical daughter cell of the zygote; ad, apical embryo domain; b, basal daughter cell of the zygote; cd, central embryo domain, hy, hypophyseal cell; su, suspensor.