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First published online 27 July 2004
doi: 10.1242/dev.01289

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Differential cytoplasmic mRNA localisation adjusts pair-rule transcription factor activity to cytoarchitecture in dipteran evolution

Simon L. Bullock^1,*, Michael Stauber^2,*, Alexander Prell², Julian R. Hughes¹, David Ish-Horowicz¹ and Urs Schmidt-Ott^2,3,

¹ Cancer Research UK, Developmental Genetics Laboratory, PO Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
² Max-Planck-Institut für Biophysikalische Chemie, Abt. Molekulare Entwicklungsbiologie, Am Fassberg 11, 37077 Göttingen, Germany
³ University of Chicago, Department of Organismal Biology and Anatomy, CLSC 921B, 920 East 58th Street, Chicago, IL 60615, USA

View larger version (60K): [in a new window]   Fig. 5. Pair-rule gene activity is reduced in egl mutant embryos. (A) Distribution of transcripts (as indicated; red) in Drosophila blastoderm embryos laid by wild-type mothers (egl+/egl+) or partial loss-of-function egl mothers (egl3e/eglWU50). mRNA alone is shown in monochrome images. Pair-rule and wg transcripts accumulate apically in wild-type embryos but are detected readily in the basal cytoplasm of mutant embryos (white arrowheads), although some apical enrichment of pair-rule transcripts is observed in most stripes (red arrowheads). The bottom panels show Run protein, which is normally apical (red arrow, left) and nuclear, but can also be detected in the basal cytoplasm in egl mutant embryos (red arrow, right). (B) The frequency of segmentation phenotypes in first instar larvae caused by inactivation of one copy of a pair-rule gene is significantly enhanced when embryos are laid by an egl mutant mother. For wild-type and mutant maternal genotypes, eve and h mutations are associated with partial deletions of even-numbered segments, whereas ftz mutations had similar effects on odd-numbered segments. We observed similar interactions with an additional h mutant allele, h31 (data not shown). The low frequency of larvae with defects in zygotically wild-type embryos typically had small notches in either even- or odd-numbered segments or partial fusion of segments. The frequency of cuticular defects caused by heterozygosity for the gap genes kni and Kr and the segment polarity gene wg are not altered significantly by the egl mutant background although the identity of segments affected by gap gene mutations differ slightly. For example, Kr1/+ normally gives defects in T3, A1 and A2 but in egl mutants defects in A3 at the expense of defects in A1 and A2 were also observed. These alterations may reflect changes in the distribution of maternal RNA determinants or reduced pair-rule activity. Numbers above the bars are total number of larvae scored. *P<0.05; ***P<0.001 (Fisher's exact test). egl is provided only maternally to the blastoderm. Experiments were conducted at 25°C, except for those involving ftz, which were carried out at 29°C, because only a few defects were seen in either genotype at 25°C. (C) Representative examples of first instar larvae with one inactive copy of h (hi22) laid by wild-type or egl mutant mothers. Red asterisks show missing regions of ventral denticle belts in abdominal segments 4 and 6 (A4 and A6). Scale bar in C: 50 µm for A (except Run protein, 30 µm); 385 µm for C.

View larger version (92K): [in a new window]   Fig. 6. Abolishing localisation of h transcripts reduces Hairy protein activity. (A-C) h transcripts (red) in wild-type (A) or transgenic (B,C) blastoderm embryos carrying one copy of a h transgene either with (B, st2-hwtD) or without (C, st2-hnlocD) a functional localisation signal under the control of the eve stripe 2 enhancer. Numbers of endogenous h stripes are indicated. Red bar indicates approximate region of enhancer activity. (D) Incidence of defects in thoracic segment 2 (T2) induced by st2-h activity in different transgenic lines. Numbers above bars indicate number of first instar larvae scored. (E) Levels of st2-h transcripts (relative to actin 5C transcripts) in different lines, as determined by real-time PCR, normalised to that of wtA. Provided RNA levels are not very high (nlocD and wtD), lines with localising st2-h transcripts have stronger phenotypic effects than those with nonlocalising transcripts, even when the localising transcript is expressed at significantly lower levels [e.g. P<0.01 for different transcript levels for wtA versus nlocB or nlocC and P=0.05 for wtB versus nlocC (t-test)]. (F-H) Lateral views of st2-h first instar larvae showing examples of (F) a normal T2 denticle belt, (G) a deletion of a ventral region of the T2 denticle belt leading to a characteristic curvature of the thorax (arrowhead points to remaining dorsal cuticle) or (H) a complete deletion of the T2 denticle belt. st2-h has been previously shown to have stronger effects ventrally (Wu et al., 2001). (I) Lateral (dorsal upwards) and (I') ventral views of st2-hnlocC/st2-hnlocC blastoderm embryos stained for ftz mRNA. In ventral views, stripes 1, 2 and 3 are shown. Anterior is towards the left. (J,J') Same views as in I,I' of st2-hwtB/st2-hwtB embryos. Ectopic h expression partially deletes ftz stripe 2 with a stronger effect ventrally. See Table S2 for quantification of ftz defects in different lines. (K-M) Confocal images of Hairy protein distribution in wild-type (K, +/+), (L) st2-hnlocC/st2-hnl°cC and (M) st2-hwtB/st2-hwtB blastoderm embryos. More Hairy protein accumulates in the nuclei between endogenous stripes 2 and 3 in st2-hwtB/st2-hwtB than it does in st2-hnlocC/st2-hnlocC, even though the former expresses significantly less st2-h mRNA. Consistent results were seen in several embryos for each genotype. Red bars in L-N show approximate regions of enhancer activity. (N) Mean fluorescent intensity/pixel within nuclei of corresponding images (data were collected with Kinetic Imaging AQM6 software). Scale bar: 50 µm in A-C; 180 µm in F-H; 130 µm in I-J', 20 µm in K-M.

View larger version (13K): [in a new window]   Fig. 1. Phylogenetic relationships of dipteran eve, h and wg genes. Newly-identified dipteran eve (A), h (B) and wg (C) sequences are more closely related to one another than to paralogous Drosophila genes. Phylogenetic distance trees were generated from ClustalW alignments of predicted protein sequences (see legend to Fig. S1 at http://dev.biologists.org/supplemental) using the Quartet Maximum-Likelihood Method of Strimmer and von Haeseler (Strimmer and von Haeseler, 1996). Numbers refer to reliability values in percent. AbdominalA (Dme-AbdA), AbdominalB (Dme-AbdB), Deadpan (Dme-Dpn), E(spl)m (Dme-HLHm), Hairy/E(spl)-related with YRPW motif (Dme-Hey), Ultrabithorax (Dme-Ubx), Dme-Wnt2, Dme-Wnt5 and Dme-Wnt6 were used as outgroups. Aga, Anopheles gambiae; Cal, Clogmia albipunctata; Cfu, Coboldia fuscipes; Dme, Drosophila melanogaster; Eba, Episyrphus balteatus; Eli, Empis livida; Hpl, Haematopota pluvialis; Mab, Megaselia abdita; Pco, Platypeza consobrina. Accession numbers: Dme-AbdA SWP, P29555; Dme-AbdB SWP, P09087; Dme-Dpn SWP, Q26263; Dme-HLHm SWP, Q01071; Dme-Eve SWP, P06602; Dme-Hairy SPTREMBL, Q95NU9; Dme-Hey SPTREMBL, Q9U9U4; Dme-Ubx SWP, P02834; Dme-Wg SPTREMBL, Q8MQP9; Dme-Wnt2 SPTREMBL, Q9V584; Dme-Wnt5 SPTREMBL, Q9VWT4; Dme-Wnt6 SPTREMBL, Q9VM26.

View larger version (64K): [in a new window]   Fig. 2. Expression and localisation of eve and h transcripts in five dipteran species at blastoderm stage. (A-J) eve and (K-T) h whole-mount in situ hybridisation showing transcript expression [(A-E,K-O) blue/purple (anterior towards the left, dorsal upwards)] and subcellular localisation [(F-J,P-T) red (apical is upwards and basal downwards in this and subsequent figures)]. Nuclear envelopes are shown in green pseudocolour (Alexa 660-wheat-germ agglutinin) in this and other figures. eve and h are found at high levels in the apical cytoplasm of Drosophila, Megaselia and Episyrphus, although in the latter species, localisation is less efficient. In Clogmia and Coboldia, these transcripts are distributed uniformly in the apicobasal axis. We identified two eve homologues in Clogmia, both of which are expressed in stripes and do not localise asymmetrically; Clogmia-eve2 is shown here. Arrowheads indicate the junction between the yolk and cytoplasm. In Coboldia and Clogmia, posteriormost stripes are established only after the onset of gastrulation (not shown). Unlike other dipteran h homologues Episyrphus-h is also expressed in the putative anlage of extra-embryonic tissue (not shown). Clogmia-h (O) is detected in a pair of anterior lateral patches and two stripes that might correspond to h stripes 1 and 6 in other species; expression in other stripe domains is weak or absent at blastoderm stages. Phylogenetic relationships of the species are shown below (Collins and Wiegmann, 2002; Yeates and Wiegmann, 1999). MYA, million years ago. Scale bar: 50 µm in F-J,P-T; 235 µm in A,K; 560 µm in B,J; 280 µm in C,M; 145 µm in D,N; 240 µm in E,O.

View larger version (80K): [in a new window]   Fig. 3. Subcellular localisation of wg transcripts in Drosophila, Coboldia and Clogmia embryos. (A-C) The segmental expression pattern of wg in extended germband embryos is conserved in Drosophila, Coboldia and Clogmia (anterior towards the left, dorsal upwards). (D-F) At blastoderm stages, wg transcripts accumulate apically in Drosophila and Coboldia, but not in Clogmia. (G-I) After gastrulation, wg transcripts accumulate apically in all three species. Localisation of wg in germband extended embryos appears to be reproducibly less efficient in Clogmia than in Coboldia and Drosophila, with a proportion of transcripts detected basally. Scale bar: 385 µm in A; 240 µm in B; 400 µm in C; 83 µm in D,F; 50 µm in E,G,I; 27 µm in H.

View larger version (29K): [in a new window]   Fig. 4. Localisation of dipteran transcripts upon injection into Drosophila blastoderm embryos mediated by Egl-dependent mRNA localisation signals. (A) Drosophila embryos injected with h transcripts from dipteran species and fixed 8-11 minutes later, showing representative examples of different efficiencies of mRNA localisation. Ten to 30 embryos were imaged for each transcript and used to categorise the extent of apical RNA enrichment. (A, bottom panels) Anopheles-h transcripts localise very efficiently upon injection (left), but localisation of this transcript is prevented by prior injection with antibodies that specifically inhibit the function of the Drosophila Egl protein (right). (B) Summary of the efficiency of localisation signals within dipteran eve, h and wg transcripts upon injection into Drosophila plotted onto the phylogenetic tree: +++, very efficient localisation; ++, efficient localisation; +, weak localisation; -, no apical enrichment. The efficiency of apical transport in this assay mirrors endogenous efficiencies of transcript localisation (compare with Figs 2 and 3). Both Clogmia-eve1 and Clogmia-eve2 fail to localise in this assay. All localising transcripts are dependent on Egl function. The occurrence of pair-rule mRNA localisation signals does not appear to be related to the absolute size of the embryo. For example, h transcripts contain localisation signals in Megaselia and Drosophila (length of the embryo is 400-500 µm), but not in Haematopota (1500 µm) or Coboldia (260 µm). Scale bar: 50 µm.

View larger version (26K): [in a new window]   Fig. 7. A model for the role of apical pair-rule mRNA localisation in Diptera. Cartoon illustrating mRNA and protein distributions in syncytial blastoderm embryos. Darker shading of nuclei represents a higher concentration of pair-rule protein. See Discussion for details. cf, cellularisation front; n, nucleus; y, yolk.