Title: Dual role for fimbriata in regulating floral homeotic genes and cell division in Antirrhinum
Abstract: Article1 November 1997free access Dual role for fimbriata in regulating floral homeotic genes and cell division in Antirrhinum Gwyneth C. Ingram Gwyneth C. Ingram Search for more papers by this author Sandra Doyle Sandra Doyle John Innes Centre, Colney Lane, Norwich, NR4 7UH UK Search for more papers by this author Rosemary Carpenter Rosemary Carpenter John Innes Centre, Colney Lane, Norwich, NR4 7UH UK Search for more papers by this author Elizabeth A. Schultz Elizabeth A. Schultz Search for more papers by this author Rüdiger Simon Rüdiger Simon Search for more papers by this author Enrico S. Coen Corresponding Author Enrico S. Coen John Innes Centre, Colney Lane, Norwich, NR4 7UH UK Search for more papers by this author Gwyneth C. Ingram Gwyneth C. Ingram Search for more papers by this author Sandra Doyle Sandra Doyle John Innes Centre, Colney Lane, Norwich, NR4 7UH UK Search for more papers by this author Rosemary Carpenter Rosemary Carpenter John Innes Centre, Colney Lane, Norwich, NR4 7UH UK Search for more papers by this author Elizabeth A. Schultz Elizabeth A. Schultz Search for more papers by this author Rüdiger Simon Rüdiger Simon Search for more papers by this author Enrico S. Coen Corresponding Author Enrico S. Coen John Innes Centre, Colney Lane, Norwich, NR4 7UH UK Search for more papers by this author Author Information Gwyneth C. Ingram2, Sandra Doyle1, Rosemary Carpenter1, Elizabeth A. Schultz3, Rüdiger Simon4 and Enrico S. Coen 1 1John Innes Centre, Colney Lane, Norwich, NR4 7UH UK 2Reproduction et Développement des Plantes, École Normale Supérieure, 46 allée d'Italie, F-69364 Lyon, cedex 01, France 3Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta, T1J 1L1 Canada 4Institut für Entwicklungsbiologie, Universität zu Köln, Gyrhofstrasse 17, 50923 Köln, Germany The EMBO Journal (1997)16:6521-6534https://doi.org/10.1093/emboj/16.21.6521 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info The fimbriata (fim) gene of Antirrhinum affects both the identity and arrangement of organs within the flower, and encodes a protein with an F-box motif. We show that FIM associates with a family of proteins, termed FAPs (FIM-associated proteins), that are closely related to human and yeast Skp1 proteins. These proteins form complexes with F-box-containing partners to promote protein degradation and cell cycle progression. The fap genes are expressed in inflorescence and floral meristems in a pattern that incorporates the domain of fim expression, supporting an in vivo role for a FIM–FAP complex. Analysis of a series of novel fim alleles shows that fim plays a key role in the activation of organ identity genes. In addition, fim acts in the regions between floral organs to specify the correct positioning and maintenance of morphological boundaries. Taking these results together, we propose that FIM–FAP complexes affect both gene expression and cell division, perhaps by promoting selective degradation of regulatory proteins. This may provide a mechanism by which morphological boundaries can be aligned with domains of gene expression during floral development. Introduction Floral meristems produce four types of organs in concentric whorls in the order sepals, petals, stamens and carpels. The identity of the organs is controlled by the combinatorial action of genes which are expressed in specific regions of the floral meristem (Schwarz-Sommer et al., 1990; Coen and Carpenter, 1993; Weigel and Meyerowitz, 1994). Mutations in these organ identity genes can be divided into three classes: in class A mutants, carpels replace sepals and stamens replace petals; in class B mutants, sepals replace petals and carpels replace stamens; in class C mutants, petals replace stamens and carpels are replaced by reiterative growth of sepals and petals. Based on these mutants, three genetic functions, a, b and c, have been proposed to specify organ identity in the combination a, ab, bc and c, in whorls 1–4 respectively (Coen and Meyerowitz, 1991). In most cases, the genes required for these functions are expressed in domains that are precisely aligned with the morphological boundaries between whorls of organs, ensuring discrete changes in organ type from whorl to whorl (Jack et al., 1992; Schwarz-Sommer et al., 1992; Bradley et al., 1993). However, the mechanisms responsible for this alignment between morphology and gene expression boundaries are unclear. A candidate gene involved in this process is the fimbriata (fim) gene of Antirrhinum, and its Arabidopsis orthologue, UFO, which affect both the identity and arrangement of organs within the flower (Simon et al., 1994; Ingram et al., 1995; Levin and Meyerowitz, 1995; Wilkinson and Haughn, 1995). The FIM protein contains a novel structural motif, the F-box, which is present in a range of proteins, including cyclin A, cyclin F and CDC4, that are involved in cell cycle control in yeast (Yochem and Byers, 1987; Zhang et al., 1993, 1995; Bai et al., 1994, 1996). Some F-box proteins, including Met30p and GRR1, act as transcriptional inhibitors and may also play a role in cell cycle control (Flick and Johnston, 1991; Thomas et al., 1995). The precise role of fim in flower development is, however, unclear. We have used a combination of molecular and genetic approaches to address this problem in Antirrhinum. Flowers of fim mutants described so far have organs consisting of petal/sepal tissue in the second whorl and petal/sepal/carpel tissue in the third whorl. In addition, mutant flowers sometimes fail to produce carpels and instead produce large numbers of mosaic organs (apical indeterminacy). These phenotypic effects can be accounted for by reduced expression of the b function gene deficiens (def) and c function gene plena (ple), suggesting that one role of fim is to promote transcription of organ identity genes (Simon et al., 1994). Consistent with this, expression of fim is detected in meristems before any organ primordia are visible, shortly before the onset of b and c organ identity gene expression. Early fim expression encompasses the presumptive b and c domains: it is first seen in a ventral (abaxial) region of the floral meristem and then in the central region, after which it resolves into a broad ring internal to the sepal primordia, consistent with its role in regulating organ identity. At later stages, however, fim expression becomes localized to the boundary regions around the base of developing petal primordia and does not overlap with most of the domain of b or c activity. This raises the question of how fim interacts with the organ identity genes at different stages of development. In addition to affecting organ identity, fim mutants also show altered organ arrangement, united growth of organs and the occasional production of meristems in the axils of floral organs (lateral indeterminacy). Thus, fim also seems to play a role in establishing organ and whorl boundaries during floral development. Here we describe two complementary approaches that have been used to study the action of fim. One involves isolating proteins that interact with FIM during flower development using the yeast two-hybrid system. The second approach involves generating a series of alleles with altered levels of fim activity. This is particularly important because previously analysed fim alleles are not nulls, obscuring the extent to which fim is required for the proper arrangement and identity of organs (Simon et al., 1994). To isolate a range of novel mutations at the fim locus, including null alleles, we exploited a weak fim allele (fim-619), which carries an insertion of the Tam3 transposon in the fim promoter. Tam3 is known to generate deletions and rearrangements in genes and can therefore be used as a localized mutagen (Martin et al., 1988; Almeida et al., 1989; Coen et al., 1989; Martin and Lister 1989; Hudson et al., 1990). Such transposon-induced derivatives can be screened for by PCR, as described in Caenorhabditis and maize (Rushforth et al., 1993; Das and Martienssen, 1995). We show that FIM associates with a family of proteins, termed FAPs (FIM-associated proteins), closely related to Skp1 proteins. In yeast and humans, Skp1 proteins interact with F-box-containing proteins, directly or indirectly, to form a complex needed for protein degradation and cell cycle progression (Zhang et al., 1995; Bai et al., 1996; Connelly and Hieter, 1996). Thus, the similarity between FIM and F-box proteins is further substantiated by their ability to interact with similar partners. The fap genes are expressed in inflorescence and floral meristems in a pattern that incorporates the domain of fim expression, supporting the conclusion that FIM forms a complex with one or more FAPs in vivo. Analysis of a series of novel fim alleles shows that fim plays a key role in the activation of the b genes def and globosa (glo), and is needed to activate expression of the c gene ple in the centre of the floral meristem at the correct time during flower development. In addition, fim acts in the regions between floral organs to specify the correct positioning and maintenance of morphological boundaries. Taking these results together, we propose that a FIM–FAP complex affects both gene expression and cell division, perhaps by promoting selective degradation of regulatory proteins. This may provide a mechanism by which morphological and gene expression boundaries can be aligned with each other during floral development. Results Molecular characterization of novel fim alleles The role of fim in the control of flower development was investigated by site-selected mutagenesis, using fim-619 as the starting material. The fim-619 allele carries a Tam3 insertion 976 bp upstream of the start of the longest fim cDNA (start of the cDNA is denoted position 0). Ten events were detected from a PCR-based screen of 4020 progeny of fim-619 (M1 generation) and the corresponding seed was sown to give 10 M2 families. Screening the plants in these families by PCR, showed that seven of the 10 events were transmitted to the M2. The three untransmitted events could either have been somatic, or present in M1 plants which did not set seed. The seven transmitted alleles, named fim-676 to fim-682, were further characterized both by PCR amplification and by cloning and sequencing the PCR products. M2 plants homozygous for the new alleles were then analysed by DNA blot hybridization. The seven alleles carried deletions extending to the right of the Tam3 by various amounts. Two of the alleles had retained the original Tam3 insertion, whereas the five other alleles had lost Tam3 (Figure 1A). These types of deletion have previously been observed as the result of plant transposon activity and have been attributed to aberrant transposition events (McClintock, 1953, 1954; Martin et al., 1988; Coen et al., 1989; Federoff, 1989; Martin and Lister 1989; Hudson et al., 1990; Robbins et al., 1990). There were no striking similarities between the sequences around the right breakpoints of the seven deletions, although the breakpoints found in the fim-676 and fim-678 alleles were separated by only 2 bp. Four of the five deletion events in which Tam3 was lost had the same left breakpoint, 11 bp away from the transposon (bases −976 to −987, Figure 1B), indicating that they may have arisen from the same type of event. Figure 1.(A) Novel deletion events detected in the fim cluster. The fim ORF is shown as a black box, and the position of Tam3 in fim-619 indicated by a large black triangle. Null alleles are indicated by asterisks. The deletions in fim-676–fim-682 are shown as open boxes. Retained Tam3 elements are indicated as black triangles. (B) Sequence analysis of novel deletion events. The Tam3 insertion in fim-619 (position −976) is indicated by a triangle (position 0 corresponds to the end of the longest fim cDNA and the fim ATG is at position +97). The 10 bp imperfect duplication to the right of Tam3 in fim-619 is double underlined and a potential MADS box protein-binding site is enclosed in a dotted box. Sequences at the break points of each deletion allele are shown, with open boxes indicating deleted nucleotides to the left of the Tam3 insertion. Deletions to the right of Tam3 are indicated by dotted lines, with the number of bp deleted marked. Null alleles are indicated by asterisks. Triangles indicate retained Tam3 elements. The fim-678 and fim-679 alleles have rearranged bases at the right breakpoint (single underlined). Where deletions extend into the fim open reading frame, amino acid sequence is shown below the nucleotide sequence. The start of the putative fim-680 protein product is shown in bold. Download figure Download PowerPoint Deletion of the fim open reading frame gives rise to a novel phenotype Plants homozygous for the deletions in fim-677, fim-679, fim-681 or fim-682 all gave a similar phenotype (Figure 2D). This was more extreme than had previously been observed for any fim allele, confirming that previously described alleles were not null. At least half of the fim open reading frame (ORF) had been deleted in these new alleles, and DNA blots confirmed that no duplications of fim were present in the genome of these plants. Thus, the phenotype shown in these plants represented the null mutant phenotype of fim. Detailed analysis of the phenotype revealed the extent to which fim was involved in: (i) organ identity; (ii) whorl integrity; and (iii) determinacy. Figure 2.Phenotypes conferred by deletions at the fim locus. Flowering spikes from wild-type (A), fim-619 (B), fim-676 (C) and the fim-677 null (D). A typical flower is shown below each spike (E, F, G and H). Each spike shows a region of abortion (arrow), probably caused by environmental fluctuation. Decreasing amounts of petal and stamen tissue with increasing allele severity are apparent from left to right, as are increases in sepal tissue production. A high degree of apical and lateral indeterminacy is visible in the basal region of the null fim spike (D, indeterminate flower indicated by an arrowhead). Download figure Download PowerPoint Changes in floral organ identity. All null fim mutants produced flowers that showed changes in organ identity that were consistent with reduced b and c activity (Figure 2D and H). Flowers usually had a first whorl of sepals, and most organs internal to these were also sepal-like. Petal tissue was rare and was only present in small localized patches on the edges of internal sepals; stamenoid tissue was never observed, even under glasshouse conditions where the phenotype was less extreme than in plants grown in the field. Carpel tissue and ovules were produced towards the centre of many flowers, especially flowers produced higher on the inflorescence spike (see later). The carpels were commonly united with adjacent organs and split or distorted. Disruption of whorl integrity. Dissection of mature flowers from fim nulls suggested that organs internal to the first whorl were not arranged in regular whorls. To determine the developmental origin of this change in phyllotaxy, early floral meristems were examined by scanning electron microscopy. Flower meristems arose in the axils of bract primordia on the periphery of the inflorescence apex. The meristems were numbered sequentially, starting with the youngest bract primordium at the top of the inflorescence apex (node 0). The development of flowers was divided into several stages as described by Carpenter et al. (1995): stage 0 (nodes 0–4, bract tongue stage); stage 1 (nodes 4–8, eye stage); stage 2 (nodes 8–10, loaf stage); stage 3 (nodes 10–12, pentagon stage); stage 4 (nodes 12–14, floritypic stage); stage 5 (nodes 15–18, petal mound stage). In null fim mutants, floral development appeared normal until about stage 4. In wild-type meristems of this stage, a whorl of five sepal primordia was visible on the periphery of the floral meristem (Figure 3A). In fim null mutants, an extra primordium was frequently produced between the two ventral (abaxial) primordia (Figure 3B, arrow). This extra primordium appeared to be initiated later than the other sepals because prior to stage 4, the shape of the floral meristem in the null mutants was pentagonal, similar to that of wild-type meristems. The ventral primordia of the first whorl developed either into sepals (as in wild-type) or, more rarely, cylindrical filamentous organs (an example is labelled with an asterisk in Figure 3H). By early stage 5 in wild-type, a whorl of petal primordia was visible as five small mounds developing alternate and internal to the sepals (Figure 3C). However, in fim null flowers at this stage, it was usually not possible to allocate internal primordia to individual whorls. In addition, ventral primordia in the first whorl were occasionally united with more internal organ primordia of the flower (Figure 3D, arrow). In some flowers, organ primordia were arranged in a symmetrical fashion on either side of the dorsoventral axis of the meristem (Figure 3F), whereas in other flowers no coherent pattern for organ initiation was apparent. Organ primordia were often shaped abnormally and united with each other, the boundaries between developing organs often being poorly defined, making identification and counting of individual organs difficult. In wild-type, the production of petal and stamen primordia was closely followed by the initiation of two central carpel primordia (stage 6, Figure 3E and G). In fim null mutants there was a delay in the appearance of further primordia after the first 10–12 had emerged (Figure 3F and H). The later primordia which eventually formed appeared to be arranged in an increasingly abnormal way, and sometimes formed a spiral towards the centre of the flower (not shown). Figure 3.The development of fim null mutant flowers. Floral meristems from wild-type and null fim-681 mutant plants are shown at nodes 13, 15, 17 and 19. At node 13 (stage 4), the dorsal region of the mutant flower appears normal but an extra primordium is visible between the ventral two organs (marked with an arrow in B). By node 15 (early stage 5) defects in the mutant are more apparent, illustrated by the extra ventral sepal which is united with a more internal organ (arrow in D, the two dorsal sepals are absent). At node 17 in wild-type, the second whorl petal primordia are small and stamen primordia are clearly visible (E, all but the most dorsal sepal removed). By contrast, at a comparable node in the fim mutant, organ primordia (probably sepals) internal to the first whorl are relatively large and those further in are hardly visible (F, all but the most dorsal first whorl organs removed). Whorl structure has also started to deteriorate. By node 19, retarded development of internal primordia is even more noticeable in the mutant (H, first whorl sepals have been left in place but have been partially dissected to reveal internal primordia) as compared with wild-type (G, all sepal primorida removed). United ventral sepals (H, arrow) and a filament structure (labelled with an asterisk in H) are also apparent in the mutant. Download figure Download PowerPoint Reduced determinacy. Various degrees of indeterminacy were observed in fim nulls. Wild-type flowers are apically determinate, producing only four whorls of organs, and laterally determinate because no meristems arise in the axils of floral organs. In fim nulls, a gradient in the degree of indeterminacy was apparent along the inflorescence spike from lower to upper regions (Figure 4). Flowers produced low down on the inflorescence spikes (flower 0–10), commonly showed both lateral and apical indeterminacy (Figure 4). The internodes of these flowers became elongated after the production of 10–24 sepals, and there was a concurrent switch to spiral phyllotaxy. Such flowers produced no carpel-like structures on the main floral axis. Some of these flowers were also laterally indeterminate, commonly having 2–12 secondary flowers, each subtended by a sepal. The secondary flowers were determinate and resembled those produced higher up on the inflorescence spike. In combination, these traits gave some early flowers many of the characteristics of an inflorescence (Figure 2D, arrowhead). Figure 4.Loss of determinacy in fim-677 flowers. Proportion of indeterminate flowers on 87 spikes from null fim mutants grown under field conditions. Flower number was counted from the lowest flower upwards, each bract being counted as a floral node. For each range of flower numbers, the proportion of indeterminate (left bar) and determinate (right bar) flowers was measured. The abortion rate in the first 15 nodes was considerably higher (up to 5%) than in concurrently grown wild-type plants (1–2%). Download figure Download PowerPoint The phenotype of upper flowers was more uniform than that of lower flowers (an example is shown in Figure 2H). They rarely showed lateral or apical indeterminacy and consequently contained fewer organs than lower flowers. In addition, the proportion of carpel tissue was considerably greater. Upper flowers were usually composed of a first whorl of sepals, followed by 4–10 sepals and then carpelloid organs in the centre of the flower. In many cases, the internal organs of the flower were united with each other and, very occasionally, flowers terminated with united carpels similar to those found in the third whorl of def or glo mutants. Phenotypes conferred by partial deletions of fim Although the fim-680 deletion extended into the ORF, its mutant phenotype was less extreme than that of the null alleles: it showed greater determinacy, more petal and carpel tissue, and less internode elongation (data not shown). This weaker phenotype might be explained by the presence of an in-frame ATG, situated 38 amino acids (aa) into the ORF; the deletion in fim-680 extended only 108 bp (36 aa) into the ORF so the mutant could have produced a protein with 38 aa missing from the N-terminus (Figure 1B). The missing N-terminus is the region of FIM that is least conserved with its homologue from Arabidopsis, UFO (Ingram et al., 1995). It is therefore probable that this part of the protein is not absolutely required for FIM activity. Two deletions gave very different phenotypes but had almost identical breakpoints in the 5′ promoter region of fim. The main difference in the structure of these alleles was whether or not they retained Tam3. In fim-678, which had lost Tam3 and 770 bp of promoter DNA, the deletion breakpoint was at −206, and in fim-676, which retained Tam3, the breakpoint was at −204. Homozygous fim-678 plants gave a wild-type phenotype, suggesting that the 770 bp 5′ region deleted in these plants was not necessary for fim transcription. However, plants homozygous for fim-676, which retained Tam3, had a mutant phenotype with sepal/petal mosaic organs in the second whorl and a third whorl comprising one to two deformed stamens together with petal/sepal/carpel mosaic organs. The phenotype of plants carrying fim-676 was more extreme than that of the fim-619 progenitor which only showed slight abnormalities in the second whorl (Figure 2). Therefore, the presence of Tam3 in the fim promoter, in combination with the loss of promoter sequence to the right of Tam3, caused considerable disruption of fim function. This effect of Tam3 could be due to transcription factors or transposase proteins binding to its ends and interfering with fim expression. Alternatively, the effects of Tam3 could be due to physical distancing of 5′ regulatory elements from the start of fim (Bradley et al., 1993; Chatterjee et al., 1996). Expression of the fim alleles The effects of the various deletions on fim expression were analysed by RNA in situ hybridizations on inflorescence apices. In wild-type, fim is first expressed in the ventral region of floral meristems and then in their centre before any floral organ primordia are visible (stage 2). The expression of fim then appears to spread outwards and, by the floritypic stage (stage 4), becomes localized as a ring around the centre of the floral meristem, adjacent to the sepal primordia (Simon et al., 1994). By stage 5, when petal primordia become visible, fim transcripts are present in domains around the base of each developing petal (Figure 5A). Throughout the rest of floral development fim remains localized at the junctions between sepals and petals, and between petals and stamens (Simon et al., 1994). Figure 5.Expression of fim def and ple in fim mutants. Expression pattern of fim, def and ple in floral meristems at around node 16–17 of wild-type (A–C), fim-676 (D–F) and the fim-677 null (G–I). Expression of fim in fim-676 (D) is considerably reduced compared with wild-type (A), although the expression domain remains essentially unchanged. No fim transcripts were detected in fim-677 (G). The domain of def expression in fim-676 (E) is similar to wild-type (B), although large areas lacking def expression are observed within some second and third whorl organs. Two such organs are visible in transverse section (arrowed in E), one showing def expression at both lateral margins and the other showing a small amount of expression at one lateral margin. A very low level of def expression can be seen near the centre of the fim-677 null floral meristem (H), although many fim null floral buds show no def expression. Expression of ple in fim-676 (F) was slightly delayed and restricted as compared with wild-type (C). Expression of ple in fim-677 (I), when present, was delayed and restricted to a small region in the centre of the floral meristem. Less than 50% of fim-677 flowers showed any ple expression at node 16. The positions of sepal (s), petal (p), stamen (st) and carpel (c) primordia in wild-type meristems are shown in (A). Scale bar is 100 μm. Download figure Download PowerPoint The deletion mutants showed reductions in fim expression which correlated with the severity of their phenotypes. The fim-678 allele, which had a 768 bp 5′ deletion and a wild-type phenotype, showed wild-type fim expression (data not shown). In contrast, fim-676, which had a similar deletion but which retained Tam3 and showed a mutant phenotype, had a reduced level of transcript, although the distribution appeared to be temporally and spatially similar to wild-type (Figure 5D). Plants homozygous for fim-680, which gave an almost null phenotype, showed a very low level of fim transcript, although the spatial domain of expression seemed normal (not shown). Detection of fim transcripts in fim-680 was delayed compared with wild-type: transcripts first became visible at around node 12 compared with node 9 in wild-type. Plants homozygous for fim-679 or fim-681, which showed a null phenotype, had very low levels of transcript with the same spatial and temporal distribution as in fim-680 (not shown). The probe used for in situ analysis extended 3′ of the deletion breakpoints in all of these alleles and could therefore detect transcripts truncated at the 5′ end. No fim transcripts were detected in plants homozygous for the null alleles fim-682 and fim-677, which had lost the entire region covered by the probe (Figure 5G). The results indicated that even when the entire 5′ region between fim and Tam3 was deleted, information conferring spatially specific expression of fim was still present. Sequences 5′ of the Tam3 excision site and/or in the 3′ of the gene therefore play an important role in fim regulation. A candidate regulatory element is a putative MADS box protein-binding site 39 bp 5′ of the Tam3 insertion site, which was not lost in any of the deletions studied (Figure 1B) (Schwarz-Sommer et al., 1990). Expression of glo and def in fim mutants The observed decrease in petal and stamen tissue in fim mutants suggested that b activity was reduced. In wild-type, the b function gene def is first expressed in the central region of floral meristems at node 10 just before the floritypic stage (stage 4). As second and third whorl primordia arise, def expression is lost from the centre of the floral meristem and becomes localized in the developing petal and stamen primordia (Schwarz-Sommer et al., 1992) (Figure 5B). Previous analysis of the fim-620 allele had shown that reduced fim activity led to a decrease and delay in the expression of def (Simon et al., 1994). A further reduction in def expression was observed in the fim nulls we generated in this study. About half of all floral meristems analysed showed no detectable def expression and in those that did show expression, it was delayed and restricted to small patches of cells in the central regions of young floral meristems. At later stages, small patches of def expression were occasionally detected in organs internal to the sepal whorl but these patches were very rare (Figure 5H). In fim null mutants, expression of another b function gene, globosa (glo), was also found to be severely diminished. The glo expression pattern in fim nulls was very similar to that of def: it was absent from many floral meristems, and very much reduced and delayed in meristems where it was detected (data not shown). The temporal and spatial expression pattern of glo is known to be closely correlated to that of def in wild-type plants. However, probing adjacent sections of fim null buds at the late floritypic stage (nodes 13–14) indicated that both def and glo expression was found in small patches of cells, but that these patches only occasionally overlapped. This indicated that these genes were being independently activated in a stochastic mann