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First published online 8 December 2004
doi: 10.1242/dev.01565

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The bHLH genes GL3 and EGL3 participate in an intercellular regulatory circuit that controls cell patterning in the Arabidopsis root epidermis

¹ Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 830 North University Avenue, Ann Arbor, MI 48109, USA
² Section of Molecular, Cell and Developmental Biology and the Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA

View larger version (63K): [in a new window]   Fig. 1. GL3 and EGL3 promoter activity in the developing root. Expression of the GL3::GUS and EGL3::GUS reporter fusions in the root epidermis of 4-day-old seedlings. The magnified images (right) show the differential cell division rate of N- and H-cell files, indicating that both reporters are preferentially expressed in developing hair cells. The GL3::GUS reporter is also expressed in the quiescent center cells in the root meristem, but this does not seem to be associated with the role of GL3 in epidermal development (C.B. and J.S., unpublished).

View larger version (107K): [in a new window]   Fig. 2. GL3 and EGL3 RNA accumulation in the developing root. Whole-mount in situ RNA hybridization of GL3 and EGL3 mRNA in 4-day-old root tips. (A) Hybridization of root tips with GL3/EGL3 sense and antisense RNA probe in wild-type (WT) seedlings. GL3/EGL3 RNAs accumulate preferentially in cells in the H position. (The staining occurs in cells with the higher cell division rate.) The panel on the far right shows that this expression pattern is maintained in epidermal clones. The corresponding sense probe does not show any signal in the root tip. (B) Hybridization of root tips with GL3/EGL3 antisense RNA probe in the wild type, gl3-2, egl3-1 and gl3-2 egl3-1 lines. This indicates that both GL3 and EGL3 RNAs accumulate in the same (H-cell) pattern.

View larger version (25K): [in a new window]   Fig. 3. GL3 and EGL3 expression and function in embryos. (A) Analysis of GL3 and EGL3 promoter activity in the root of mature wild-type (WT) embryos using GL3::GUS and EGL3::GUS reporter lines. GL3 and EGL3 are expressed in the same H-cell pattern in the embryonic root as in the post-embryonic root. (B) Expression of the GL2::GUS and CPC::GUS reporters in hypocotyl and root epidermis of mature wild-type and gl3 egl3 mutant embryos. GL2 and CPC promoter activity is reduced in the gl3 egl3 mutant in both hypocotyl and root (except that no CPC::GUS activity is detected in the embryonic root epidermis in either line).

View larger version (67K): [in a new window]   Fig. 4. Regulation of the EGL3::GUS expression pattern. Expression of the EGL3::GUS reporter in the developing root epidermis of 4-day-old seedlings in mutant backgrounds and transgenic overexpressing lines. (A) EGL3 promoter activity is expanded in the epidermis of the wer and ttg lines, but unaffected by the gl2 and rhd6 backgrounds. (B) EGL3 promoter activity is reduced in the cpc try line (but not significantly affected in either single mutant), and it is expanded in the epidermis of the 35S::CPC and 35S::TRY backgrounds. (C) EGL3 promoter activity is expanded in the epidermis of the gl3 egl3 line (but not in either single mutant), and it is reduced in the 35S::GL3 and 35S::EGL3 backgrounds.

View larger version (43K): [in a new window]   Fig. 5. Regulation of the GL3::GUS expression pattern. Expression of the GL3::GUS reporter in the developing root epidermis of 4-day-old seedlings in various mutant backgrounds and transgenic overexpressing lines. Ectopic GL3 promoter activity is found in the wer, gl3 egl3 and 35S::CPC background. Reduced GL3 promoter activity is present in the cpc try mutant.

View larger version (67K): [in a new window]   Fig. 6. GL3 and EGL3 RNA accumulation in mutant and overexpression lines. Whole-mount in situ RNA hybridization of GL3 and EGL3 mRNA in 4-day-old root tips in wild-type (WT), wer, cpc try and 35S::CPC background. GL3/EGL3 RNAs accumulate throughout the epidermis in wer and 35S::CPC seedling roots, while no GL3/EGL3 RNAs were detected in cpc try root epidermis.

View larger version (60K): [in a new window]   Fig. 7. Localization of a GL3-YFP translation fusion in the root epidermis. Expression of a GL3::GL3-YFP construct in the developing root epidermis of 4-day-old gl3-2 mutant seedlings. First three panels: wide-field fluorescence microscope images. Stars indicate cells files composed of developing non-hair cells, as determined by tracing the files to the mature region of the root. Right-most panel: confocal microscope image (cell walls were counterstained with propidium iodide (red signal) to enhance visualization). The GL3 fusion protein accumulates predominantly in the nuclei of the developing non-hair cells, as apparent from their lower cell division rate and paired nature of these cell files. Consistent with the GL3::GUS expression pattern (Fig. 1), the GL3 fusion protein also accumulates in the quiescent center cells of the root meristem.

View larger version (65K): [in a new window]   Fig. 8. Proposed model for the involvement of GL3/EGL3 in a novel intercellular regulatory circuit. A WER/GL3/EGL3/TTG complex forms in cells in the N position and promotes expression of GL2 and CPC. Accumulation of GL2 in the N-position leads to the specification of the non-hair cell fate, while CPC/(TRY) moves laterally to the neighboring cell in the H position to form the inactive complex CPC/GL3/EGL3/TTG, which prevents expression of GL2 in the future hair cell. The presence of CPC/(TRY) in the H position also leads to activation of GL3 and EGL3 expression. In a lateral feedback loop, GL3/(EGL3) protein moves to the N cell to participate in the WER/GL3/EGL3/TTG complex, which activates GL2 and CPC and inhibits expression of GL3 and EGL3. See text for further discussion. Unbroken lines indicate gene transcription regulation; broken lines indicate protein movement; dotted lines indicate little/no transcription regulation. Proteins shown in white are at a low concentration.