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doi: 10.1242/10.1242/dev.00493

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Epidermal patterning genes are active during embryogenesis in Arabidopsis

Department of Cell and Developmental Biology, John Innes Centre, Norwich, NR4 7UH, UK

View larger version (121K): [in a new window]   Fig. 1. In situ hybridisation of GL2 mRNA in developing wild-type embryos. In the schematics in B, D and F, RNA localisation is indicated in orange. (A) Serial longitudinal sections through a heart stage embryo. Weak expression of GL2 mRNA is detected at the basal end of the embryo, in the protodermal layer, in alternate sections (arrows). Inset: a longitudinal section hybridised with GL2 sense probe shows no signal. (B) Schematic representation of the GL2 expression pattern detected in one of the sections in A. (C) Transverse sections through the torpedo stage embryo. From the left to the right of the panel the sections displayed were taken from: the root pole at the base of the embryo, the root close to the root pole, the root close to the hypocotyl, the hypocotyl. At the root pole, GL2 mRNA is present in the root cap, lateral root cap and throughout the epidermis. In the hypocotyl, GL2 mRNA is present in epidermal cells that overlie a single cortical cell. Inset: section hybridised with GL2 sense probe. (D) A schematic representation of the longitudinal organisation of the embryo at torpedo stage and the radial organisation in the embryonic root (a) and hypocotyl (b) as indicated. (E) Transverse sections through the mature stage embryo. From the left to the right of the panel the sections displayed were taken from: the root pole at the base of the embryo, the root close to the root pole, the root close to the hypocotyl, the hypocotyl. In the root, GL2 is expressed only in those cells that overlie the walls of single cortical cells that will develop into non-hair epidermal cells in the seedling. In the hypocotyl GL2 is only expressed in epidermal cells that will become elongated, non-stomatal cells overlying the walls of single cortical cells. Inset: section hybridised with GL2 sense probe. (F) A schematic representation of the longitudinal organisation of the embryo at the mature stage and the radial organisation of the root (a) and the hypocotyl (b) as indicated. Scale bars: 40 µm.

View larger version (96K): [in a new window]   Fig. 2. Localisation of WER mRNA in developing wild-type embryos. (A) Longitudinal sections through a globular (a) and a heart (b) stage embryo and schematic cellular organisation. No expression of GL2 mRNA is detected in either embryonic stage. Inset: a longitudinal section hybridised with WER sense probe shows no signal. (B-E) Transverse sections through embryos at different developmental stages hybridised with WER antisense RNA probe, and schematic representations of the cellular organisation of root and hypocotyl. The localisation of mRNA is indicated in orange on these schematics. From the left to the right of B and D the sections displayed were taken from: the root pole at the base of the embryo, the root close to the root pole, the root close to the hypocotyl, the hypocotyl. (B) Torpedo stage. WER mRNA is detected in lateral root cap and epidermis at the root pole. In the hypocotyl WER expression is detected throughout the epidermal layer. Inset: section hybridised with WER sense probe. (C) A schematic representation of the longitudinal organisation of the embryo at torpedo stage and the radial organisation in the embryonic root (a) and hypocotyl (b) as indicated. (D) Mature stage. WER expression was not detected in either the root or the hypocotyl. Inset: section hybridised with a WER sense probe. (E) A schematic representation of the longitudinal organisation of the embryo at the mature stage and the radial organisation of the embryonic root (a) and hypocotyl (b) as indicated. Scale bars: 40 µm.

View larger version (94K): [in a new window]   Fig. 3. Pattern of CPC mRNA localisation in wild-type embryos. (A) Longitudinal sections through a globular (a), an early-heart (b) and a mid-heart (c) stage embryo and schematic cellular organisation. No expression of CPC mRNA is detected in any embryonic stage. Inset: a longitudinal section hybridised with CPC sense probe shows no signal. (B-E) Transverse sections through embryos at different developmental stages probed with CPC antisense RNA and schematic representation of the cellular organisation of root and hypocotyl. The localisation of mRNA is indicated in orange on these schematics. From the left to the right of B and D the sections displayed were taken from: the root pole at the base of the embryo, the root close to the root pole, the root close to the hypocotyl, the hypocotyl. (B) Torpedo stage. CPC mRNA is present throughout the root. In the hypocotyl CPC mRNA is present throughout the epidermal and cortical layers. Inset: a transverse section hybridised with CPC sense probe shows no signal. (C) A schematic representation of the longitudinal organisation of the embryo at torpedo stage and the radial organisation in the embryonic root (a) and hypocotyl (b) as indicated. (D) Mature stage. CPC is preferentially expressed in root cap and lateral root cap cells in the root. In the hypocotyl CPC expression is confined to the vascular tissues. Inset: transverse section hybridised with CPC sense probe. (E) A schematic representation of the longitudinal organisation of the embryo at mature stage and the radial organisation in the embryonic root (a) and hypocotyl (b) as indicated. Scale bars: 40 µm.

View larger version (95K): [in a new window]   Fig. 4. Localisation of GL2, WER and CPC mRNA in transverse sections through mutant embryos at different developmental stages. From the left to the right of A,C,K,M the sections displayed were taken from: the root pole at the base of the embryo, the root close to the root pole, the root close to the hypocotyl, the hypocotyl. E and G are consecutive sections throughout heart stage embryos. (A) GL2 expression in wer mature embryo. No GL2 mRNA was detected in either the root or the hypocotyl. Inset: transverse section hybridised with GL2 sense probe. (B) Schematic representations of the cellular organisation in transverse sections of the root (a) and the hypocotyl (b). (C) WER expression in gl2 mature embryo. WER mRNA is detected in every epidermal and lateral root cap cell in the root and throughout the epidermal layer in the hypocotyl. Inset: transverse section hybridised with WER sense probe. (D) Schematic representations of the cellular organisation in transverse sections of the root (a) and the hypocotyl (b) and the localisation of WER mRNA (orange). (E) GL2 expression in wer heart stage embryo. No expression of GL2 mRNA is detected. Inset: a longitudinal section hybridised with GL2 sense probe shows no signal. (F) Schematic representation of the cellular organisation of one of the sections in E. (G) GL2 expression in cpc heart stage embryo. GL2 mRNA is detected in protodermal cells both in the centre and at the basal end of the embryo (arrows). Inset: transverse section hybridised with GL2 sense probe. (H) Schematic representation of the GL2 expression pattern (orange) detected in one of the sections in G. (I) GL2 expression in cpc mature embryo. From the left to the right of the panel the sections displayed were taken from: the root pole at the base of the embryo, the root close to the root pole, the hypocotyl. GL2 mRNA is detected throughout the root cap, lateral root cap and epidermal layers in the zone above the initials. In the hypocotyl GL2 mRNA is detected in epidermal cells that overlie single cortical cells, as it is in the wild-type embryo. Inset: transverse section hybridised with GL2 sense probe. (J) Schematic representations of the cellular organisation in transverse sections of the root (a) and the hypocotyl (b) and the localisation of GL2 mRNA (in orange). (K) CPC expression in gl2 mature embryo. CPC mRNA is present in lateral root cap of the root and in the vascular precursor cells in the hypocotyl. Inset: transverse section hybridised with CPC sense probe. (L) Schematic representations of the cellular organisation in transverse sections of the root (a) and the hypocotyl (b) and the localisation of CPC mRNA (in orange). (M) CPC expression in gl2 torpedo stage embryo. CPC mRNA is present in lateral root cap of the root and in the vascular precursor cells in the hypocotyl. Inset: transverse section hybridised with CPC sense probe. (N) Schematic representations of the cellular organisation in transverse sections of the root (a) and the hypocotyl (b) and the localisation of CPC mRNA (in orange). (O) CPC expression in wer embryo. No CPC mRNA is detected in transverse sections in the root (a) and hypocotyl (b). (P) WER expression in cpc embryo. No WER mRNA is detected in transverse sections in the root (a) and hypocotyl (b); inset, section hybridised with WER sense probe. Scale bars: 40 µm.

View larger version (107K): [in a new window]   Fig. 5. Pattern of GFP expression in J2301 enhancer trap embryos. (A) Confocal images showing the GFP expression in J2301 embryos dissected from the seed coats at different developmental stages. From left to right: heart stage (a), early, mid and late torpedo stage (b,c,d) and bent cotyledons stage (e). Arrowheads point to cells expressing GFP. (B) In situ hybridisation of GFP mRNA in transverse sections of a late torpedo stage embryo of the J2301 enhancer trap line. From the left to the right the sections displayed were taken from: the root pole at the base of the embryo, the root close to the root pole, the root close to the hypocotyl, the hypocotyl. GFP is expressed in lateral root cap cells in the root and in epidermal cells in the hypocotyl. (C) Schematic representations of the cellular organisation in transverse sections of the root (a) and the hypocotyl (b) and the localisation of mRNA (in orange). Scale bars: 40 µm.

View larger version (66K): [in a new window]   Fig. 6. Embryonic expression of the J2301 enhancer trap in wild-type, cpc, gl2 and wer mutants. (A) Confocal images showing the absence of GFP expression in gl2 mutant embryos carrying the J2301 enhancer trap dissected from the seed coat at different developmental stages. Scale bar, 60 µm. The dark green colour in whole embryos does not represent GFP expression. The image was obtained using and open iris and high gain during the collection of the optical sections with the confocal microscope in order to visualise the embryos. (B-G) Detection of GFP expression at the basal pole of mature wild-type, cpc and gl2 mutant embryos carrying the J2301 enhancer trap using confocal microscopy (B,D,F) and by in situ hybridisation (C,E,G). (B) In wild type, strong GFP expression was detected in cells at the basal end of the embryo, which in median longitudinal section (C) was shown to be precisely located in lateral root cap cells. In cpc embryos (D,E) and gl2 embryos (F,G) GFP expression was not detected. Scale bars: 60 µm.

View larger version (37K): [in a new window]   Fig. 7. Schematic representation of the expression of patterning genes in the developing root during embryogenesis (upper row) and the proposed model for their transcriptional regulation (lower row). Cortex is shown in yellow, epidermis in white and lateral root cap in grey. (Upper row) GL2 is first expressed in the heart stage embryo in a subset of cells in the protoderm. By the torpedo stage, GL2 and WER are expressed in all cells of the epidermis and CPC is expressed in the cortex, epidermis and lateral root cap (C). In mature stage embryos GL2 is expressed in the future non-hair cells and CPC is expressed in the root cap. (Lower row) WER positively regulates GL2 transcription in heart stage embryos. At the torpedo stage WER positively regulates GL2 transcription while CPC negatively regulates GL2 transcription in future hair cells, which overlie the cleft between two cortical cells. In the mature embryo GL2 negatively regulates WER transcription that in turn positively regulates CPC transcription. CPC is required for preferential accumulation of GL2 in future non-hair cells.