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The Handlebars gene is required with Phantastica for dorsoventral asymmetry of organs and for stem cell activity in Antirrhinum

Richard Waites^*, and Andrew Hudson

University of Edinburgh, Institute of Cell and Molecular Biology, King’s Buildings, Mayfield Road, Edinburgh, EH9 3JH, UK
Present address: Department of Biology, University of York, PO Box 373, York, YO10 5YW, UK

View larger version (80K): [in a new window]   Fig. 1. Effects of phan and hb mutations on embryonic development. (A) Wild-type and phan mutant embryos form functional shoot apical meristems and dorsoventrally asymmetric cotyledons whereas phan hb double mutant seedlings produce ventralised cotyledons and either lack a functional SAM (left) or produce a SAM that terminates in formation of an ectopic leaf (right). (B) Scanning laser confocal micrographs of SAMs of wild-type seedlings (left) with a layered structure and recognisable central zone (CZ) which is lacking in phan hb double mutant seedlings (right).

View larger version (41K): [in a new window]   Fig. 2. Expression of the Amstm gene. In situ hybridisation was used to detect expression of the Amstm gene in (A) wild-type seedlings, (B) phan hb double mutant seedlings, (C) wild-type vegetative apices, (D) vegetative apices of phan single mutants, and phan hb double mutant apices that were still active (E) or had terminated growth (F). Arrowheads indicate expression in the axils of leaves.

View larger version (128K): [in a new window]   Fig. 3. Effects of phan and hb mutations on apical meristems. (A) Wild-type seedling with a functional SAM (arrow) and phan hb double mutant seedlings with adventitious SAMs derived from (B) the hypocotyl or (C) needle-like cotyledons and hypocotyl. (D,E) Wild-type seedlings produce similar adventitious SAMs following ablation of the original SAM. (F) Scanning laser confocal micrographs of a phan hb double mutant seedling with an adventitious SAM derived from the cotyledon (c; upper inset) and a non-functional SAM at the hypocotyl apex (h; lower inset). (G-J) Comparison of SAMs from (G) wild type, (H) phan single mutant, (I) phan hb double mutants in which the SAM is still active, or (J) has recently terminated in leaf formation. (K-M) The leaf axils of (K) wild type, (L) a phan single mutant and (M) phan hb double mutant pet, leaf petiole; ax, axillary meristem. (N) A flower of the phan hb double mutant consisting of sepals (sep) surrounding organs of uncertain identity.

View larger version (51K): [in a new window]   Fig. 4. Effects of phan and hb mutations on leaf dorsoventrality. (A) Shoots of wild type, the phan single mutant and a phan hb double mutant. (B) Transverse sections through the leaf midrib (lf) and petiole (pet) of wild type showing asymmetry along the dorsoventral axis (top to bottom of page). Needle-like leaves of phan single and phan hb double mutants (right) both lack dorsoventral asymmetry.

View larger version (38K): [in a new window]   Fig. 5. An allelic series of phan mutations. (A) Shoots and flowers of Phan+ (wild type), and three mutants grown at the semi-permissive temperature of 20°C. (B) The phan-250 mutant allele carries an uncharacterised insertion (grey triangle) within the 5'-UTR (unfilled box) and phan-249 contains an insertion of Tam2 (black triangle) in the coding region 3' to the conserved myb region (striped box). The phan-164 mutation carries an insertion of Tam 7 (grey triangle) 45 bp further downstream. The arrowheads labelled A, B and C denote the positions of primers used in the expression analysis shown in D. (C) Amino acid sequence differences between the protein products of the phan-164 and phan-249 alleles compared to wild type, as inferred from cDNA sequences. Phan sequences are shown in white type, and novel amino acids encoded by the transposons in black. (D) Phan mRNA expression in Phan+ (wild type) and plants homozygous for different phan mutant alleles, assayed by RT-PCR using primers A and B, or by 3'-RACE using primer C and an oligonucleotide complementary to the poly(A) tail.

View larger version (65K): [in a new window]   Fig. 6. Hb is required for floral asymmetry. (A) The wild-type corolla contains two dorsal petal lobes (d), two lateral lobes (l) and one ventral lobe (v) shown here detached from the corolla tube with their dorsal surfaces uppermost. Each type of petal lobe has a characteristically asymmetric shape and distribution of pigment. The corresponding petal lobes of hb single mutants show reduced asymmetry in both shape (stars) and pigmentation (arrows). (B) In the wild-type corolla tube (viewed from below), the ventral petal is characterised by a bulge in its proximal region and two stripes of pale pigmentation mark its boundaries with lateral petal. In hb single mutants, the bulge extends laterally. Reduced activity of Cyc or Rad genes has a similar effect (data not shown).

View larger version (64K): [in a new window]   Fig. 7. Hb acts independently of other floral asymmetry genes. Comparisons of floral asymmetry. (A) Wild type. (B) hb single mutant. (C) phan single mutant. (D) rad and rad hb mutants. (E) dich and dich hb mutants, upper petal lobes of dich hb double mutants are more symmetrical than either dich or hb single mutant. (F) cyc and cyc hb mutants. (G) div/+, div/+ hb, div/div, and div/div hb mutants – div mutations are semi-dominant.