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First published online December 7, 2008
doi: 10.1242/10.1242/dev.030270

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Evidence for a composite anterior determinant in the hover fly Episyrphus balteatus (Syrphidae), a cyclorrhaphan fly with an anterodorsal serosa anlage

University of Chicago, Department of Organismal Biology and Anatomy, CLSC 921B, 920 E. 58th Street, Chicago, IL 60637, USA.

View larger version (42K): [in this window] [in a new window]   Fig. 1. Phylogenetic occurrence of bicoid and anterodorsal serosa anlage of Episyrphus. Taxa with bicoid are marked by a filled circle. The serosa anlage was labeled by in situ hybridization using an Eba-zen probe. The same embryo is shown in lateral (left) and dorsal (right) views with the anterior towards the left.

View larger version (58K): [in this window] [in a new window]   Fig. 2. Expression of Eba-eve, Eba-h mRNA and Engrailed in Episyrphus. (A-B') Eba-eve expression at (A,A') and shortly after (B,B') the beginning of gastrulation. Note the clearance of Eba-eve expression along the dorsal midline (arrow in A') and the interstripes (arrowheads in A' and B'). (C-D') Eba-h expression at (C,C') and shortly after (D,D') the beginning of gastrulation. Note the gap in Eba-h expression in the serosa anlage (arrow in C') and in the eighth stripe (arrows in D,D'). (E-F') Engrailed pattern in Episyrphus embryos as detected by 4D9 antibody at the extended germband stage (E,E') and after germband retraction (F,F'). Expression in the clypeolabrum (Lr), the ocular segment (Oc), the antennal segment (Ant), the intercalary segment (Ic), the mandibular segment (Md), the maxillar segment (Mx), the labial segment (La), T1, A1 and A8 is indicated. Each embryo is shown with anterior towards the left in lateral (A-F) and dorsal (A'-D') or ventral (E'-F') view. Scale bar in F': 200 µm for A,A',C-E'; 240 µm for B,B'; 190 µm for F,F'.

View larger version (97K): [in this window] [in a new window]   Fig. 3. Cuticle of the first-instar larva. (A-C) Cuticles in lateral (A), dorsal (B) and ventral (C) view in dark-field optics. Filzkörper (fk) within the posterior spiracles (arrowhead in A), and the reduced denticle field of A8 (arrowhead in C) posterior of the presumptive anal slit are indicated. Lateral denticle doublets (arrows in A), which are characteristic for abdominal segments A1 to A7, are present. Thoracic segments T2 and T3 (arrows in B) and abdominal segments A1 to A7 share a single row of large dorsal denticles. T3 has a unique row of large ventral denticles (double arrow in C; the dorsal denticle row, which is out of focus, is marked by a gray arrow). (D-H) Magnified dorsal (D,E,G,H) and lateral (F) views of the antennomaxillary complex (amso) and cephalopharyngeal skeleton in phase contrast. The antennomaxillary complex includes the maxillary (mso) and the antennal sense organ (aso). The cephalopharyngeal skeleton includes a median tooth (mt), mouth hooks (mh), dorsal and ventral cephalopharyngeal plates (dcp, vcp), H-piece (Hp), neck clasps (nc), and Lateralgräten (lg). Anterior is towards the left. Scale bar in H: 180 µm for A; 200 µm for B,C; 44 µm for D-F; 27 µm for G,H.

View larger version (120K): [in this window] [in a new window]   Fig. 4. Expression of Eba-hb, Eba-nos, Eba-cad and Eba-otd mRNA. (A-H) Eba-hb expression during subsequent blastoderm (A-F) and gastrulation stages (G-H). Note the posterior boundary of the anterior domain (arrowheads in A-C) and extra-embryonic expression (arrows in D-G). (I-L) Eba-nos expression before (I), at the beginning of (J) and during (K) cellularization, as well as in the extended germband (L). Within pole cells, the transcripts were predominantly localized in the posterior half (J'). (M-S') Eba-cad expression before blastoderm formation (M), during consecutive blastoderm stages (N-Q), at the beginning of gastrulation (R) and during germband retraction (S,S'). Note the anterior boundary of the early zygotic expression domain (triangle in N). (T-X') Eba-otd expression at the onset of cellularization (T), during cellularization (U), at the onset of gastrulation (V,V') and during germband extension (W-X'). Note clearance along the dorsal midline of the blastoderm (arrows in U,V'), and expression along the ventral midline (arrows in W,W') and in the developing nervous system (X,X'). Embryos are shown with anterior towards the left in lateral view, except in D-G,J',S',V' and W',X', which are dorsal and ventral views, respectively.

View larger version (106K): [in this window] [in a new window]   Fig. 5. Ectopic Eba-nos activity suppresses anterior development. (A) Comparison of bicoid (bcd) and hunchback (hb) NREs with NRE-like sequences in the 3'UTRs of Eba-hb and Eba-otd.(B-G) Phenotypes caused by double-injection of Eba-nos mRNA at the anterior pole (0% EL) and 25% EL. Note the complete absence of anterior cuticular markers (B; triangles mark the Filzkörper). Note also the absence of Eba-otd expression during blastoderm cellularization (C) and Eba-hb expression prior to (D) but not shortly after (E,E') the onset of cellularization, when expression resumes in an anterior cap. Extra-embryonic Eba-hb expression (arrow in E') is confined to mid-dorsal blastoderm. Early Eba-cad expression (F) is not affected, but Eba-cad transcript degradation in older blastoderm embryos is perturbed (G). Anterior is towards the left. Embryos are shown in lateral (C-G) or dorsal (E') view. Scale bar in B: 200 µm.

View larger version (79K): [in this window] [in a new window]   Fig. 6. Functional analysis of Eba-hb and Eba-otd. (A-E') Eba-hb RNAi phenotype. Overview of the cuticular phenotype in ventral view (A), details of the head cuticle in dorsal view (B-D) and Engrailed expression after germband retraction in lateral (E) and ventral view (E'). Note that A1 retains the Engrailed-positive cell in the anterior compartment, which is characteristic for segments A1 to A7 (arrowheads in E). (F-K) Eba-otd RNAi phenotype. Overview of the cuticular phenotype in ventral view (F), details of the head cuticle in dorsal view (G-I), and Engrailed expression during (J,J') and after (K) germband retraction in dorsolateral (J) and lateral (J',K) view. Note the dorsal position of the stomodeum (St). (L) Double RNAi against Eba-hb and Eba-otd. The cuticular phenotype, shown here in ventral view, is essentially additive. (M) Ectopic anterior Eba-otd activity represses anterior expression of Eba-zen. The embryo (dorsal view) had been injected with Eba-otd mRNA at 0% EL and was stained after attaining the cellular blastoderm stage with an in situ probe against Eba-zen. In all panels anterior is towards the left. Ant, antennal segment; amso, antennomaxillary complex; aso, antennal sense organ; dcp, dorsal cephalopharyngeal plate; Hp, H-piece; La, labial segment; Lr, clypeolabrum; lc, intercalary segment; lg, Lateralgräten; Md, mandibular segment; Mx, maxillar segment; mh, mouth hooks; mso, maxillary sense organ; mt, median tooth; nc, neck clasps; Oc, ocular segment; vcp, ventral cephalopharyngeal plate. Scale bar in M: 200 µm for A,E,E'; 73 µm for B; 36 µm for C,D; 342 µm for F; 57 µm for G; 117 µm for H,I; 186 µm for J; 82 µm for J'; 211 µm for K; 285 µm for L; 22 µm for M.

View larger version (83K): [in this window] [in a new window]   Fig. 7. Functional analysis of Eba-cad. (A-H) Strong Eba-cad RNAi phenotypes. (A) Cuticle in ventral view. (B-D) Magnified head structures. (E-F') Representative Engrailed patterns in embryos of an intermediate (E) and a strong phenotype (F,F'). (G,H) Representative Eba-eve (G) and Eba-h (H) expression patterns in blastoderm embryos during cellularization. (I,J) Cuticular phenotypes after double-injection of Eba-cad mRNA at 0% and 25% EL (I) or single injection at the posterior pole (100% EL; J). (K,L) Cuticle showing a strong phenotype after double RNAi against Eba-cad and Eba-nos (K). Note mid-ventral symmetry plane (arrows) with a reversal of denticle polarity (arrowheads) and abdominal denticles (A), as well as sclerotized material and anterior sense organs at the posterior pole (L). Anterior is towards the left. Views are lateral (E-J), ventral (A-D,F',K,L) and dorsal (G',H'). Ant, antennal segment; amso, antennomaxillary complex; aso, antennal sense organ; dcp, dorsal cephalopharyngeal plate; La, labial segment; Md, mandibular segment; Mx, maxillar segment; mh, mouth hooks; mso, maxillary sense organ; mt, median tooth; nc, neck clasps. Scale bar in L: 100 µm for A,B; 31 µm for C,D; 187 µm for E-F'; 178 µm for G,H; 205 µm for I; 160 µm for J; 155 µm for K; 35 µm for L.

View larger version (6K): [in this window] [in a new window]   Fig. 8. Model of genetic factors and interactions that determine anterior development in Episyrphus. Factor 1 (F1) includes anteriorly enriched Nanos-responsive mRNA and controls the activation of Eba-otd (0-30% EL) and early (pre-cellularization) Eba-hb (0-90% EL) transcription. Factor 2 (F2) does not respond to ectopic Eba-nos activity and mediates restricted zygotic expression of Eba-hb (0-30% EL) and Eba-cad (20-100% EL). Factor 2 is sufficient to repress posterior development at the anterior pole. In Drosophila, the functions of Factor 1 and 2 are both performed by bicoid.

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