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doi: 10.1242/10.1242/dev.00242

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Functional subdivision of trunk visceral mesoderm parasegments in Drosophila is required for gut and trachea development

Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

View larger version (51K): [in a new window]   Fig. 1. Metameric hh expression in the trunk visceral mesoderm. hh expressions in wild-type embryos at late stage 11 (A,B,D) or mid-stage 12 (C) are shown. Lateral views of whole embryos (A,B) and enlarged VM-PSs (C,D) stained as indicated. Anterior is leftwards in this and following figures. In addition, except for Figs 4, 6, 7, white arrows indicate VM. PSN and vN (N, numeral), respectively, indicate ectodermal parasegment number and VM-PS number. (A) hh RNA (A1, red) and VM-hh-lacZ (A2, green) expression. (A3) Merged picture. (B) HH protein expression. (C) PTC (C1,2, red) and VM-hh-lacZ (C2, green) expression. Arrowheads, PTC signals in VM. Asterisks, ectodermal PTC signals. (D) hh RNA (D1, red) and ftz-lacZ (D2, green). (D3) Merged picture. hh RNA and ftz-lacZ expression indicate that VM-hh expression occurs in the anterior terminal region of each VM-PS. Interpretation of D1-3 is shown at the bottom.

View larger version (52K): [in a new window]   Fig. 2. VM-PS subdivision by metameric gene expression. Lateral views of wild-type whole embryos (A-D) and enlarged VM-PSs at late stage 11 to early stage 12 (E-H) stained as indicated. (A) bnl. (B)TIN. (C) bap-lacZ. (D) CON. (E-H) Vertical broken lines indicate gene expression boundaries. Interpretation of each picture is shown below. E3-H3 are merges of E1-H1 and E2-H2. (E) CON and VM-hh-lacZ are colored in red and green, respectively. (F-H) TIN is colored in red. bap-lacZ (F), bnl RNA (G) and VM-hh-lacZ (H) are colored in green. (I) Model of VM-PS subdivision. Asterisks indicate regions present in abdominal VM-PSs but absent from thoracic VM-PSs (VM-PS3-5). a and p, respectively, indicate a and p regions that form the tin/bnl/bap domain at the VM-PS border.

View larger version (85K): [in a new window]   Fig. 3. Regulation of hh and tin expression and VM cell number by HH and WG. Lateral (A-K,N,O), ventrolateral (L,M) and ventral (P-S) views of early stage 11 (L,M), mid stage 11 (P-S) and late stage 11 to early stage 12 embryos (A-K,N,O) stained as indicated on various genetic backgrounds. (A,H,L,N) Wild type, (B,K) hh13C, (C,I) wgCX4, (D,J) UAS-wg/24B-GAL, (E) twi-GAL4/+; UAS-hh, wgCX4/+, wgCX4, (F,G) twi-GAL4/+; UAS-wg, hh13C/+, hh13C, (M,O) UAS-hh/+; 24B-GAL4/+. (B,F) Note hh13C is a point mutation, which enabled us to monitor hh RNA expression. (F,G) Note in hh13C embryo misexpressing wg, TIN expression in G shows VM is at least partially formed. VM is marked with CON (red; E1) or broken white lines (E2,L,M). Arrowheads in G and H indicate cardiac signals mostly out of focus. (N,O) Drawings in the lower margin show panel interpretation. (P-S) Embryos stained for FAS3. (P) Wild type, (Q) wgCX, (R) hh13C and (S) hhts (hh9K/hh13C) embryos shifted-up from early stage 10 (4.5 AEL) onwards. Broken white lines indicate VM progenitors. Average cell numbers per VM-PS are shown below. meso in E-G,M,O indicates that the expression of target genes is driven by GAL4 drivers. Arrows in A-K indicate VM.

View larger version (71K): [in a new window]   Fig. 4. Determination of the critical period sensitive to HH and WG signals, using hhts (hh9K/hh13C) and wgts (wgIL114) mutants. (A,F) Black boxes, permissive temperature (18°C); white boxes, non-permissive temperature (29°C). Developmental time in this and subsequent sections is shown by time after egg laying (AEL) at 25°C. tin, con and VM-hh expression was examined at late stage 11 to early stage 12. +, normal or virtually normal expression; -, no expression; a), abnormal restriction of expression area (see L-N). Horizontal lines show metameric expression of VM-hh (yellow), con (red), VM-bnl (blue) and tin (green) in wild type. Gray bars associated with thick gray broken lines, deduced critical periods when VM-hh, con and tin are sensitive to HH (A) or WG (F). (B-E,G-N) Examples of shift-up/down effects on expression of three VM-metameric genes. hhts (B-E) and wgts (G-N) embryos were shifted down (D,J) or up (B,C,E,G-I,L-N) at indicated time AEL. (B-E,G-J) Lateral views of late stage 11 to early stage 12 embryos stained as indicated. (K-N) Ventral views of mid-stage 11 VM. Asterisks indicate expression of TIN (K,L) and bnl (M). (K) Wild type. a and p indicate a and p regions of VM-PSs, respectively. (L,M) Elimination of wg activity from early stage 11 on caused TIN/bnl expression only in posterior halves of the presumptive expression domains, which correspond to the anterior terminal region of each VM-PS. (N) Elimination of TIN activity from mid-stage 10 in response to absence of wg activity from mid-stage 10. (O) Ventrolateral views of wild-type early stage 11 VM. O2 is slightly younger than O3. Asterisks indicate tin RNA expression. Arrows in B-E,G-J indicate VM; arrows in K-O3 indicate VM-PS boundaries.

View larger version (39K): [in a new window]   Fig. 8. Model of transcriptional regulation of VM-metameric genes during stages 10-11. Ventrolateral view of VM is shown schematically. Gray strip, ectodermal cell layer. Green and brown-outlined boxes, ectodermal WG and HH sources, respectively. Blue horizontal arrows in the top diagram show the posterior expansion of the VM competent region; black arrows indicate activation; black-T-shaped-lines indicate repression. WG and HH susceptible regions are shown by colored lines along the future VM. tin/bnl/bap (green), VM-hh (yellow) and con (crimson) expression are indicated. Reduced con expression at the anterior end of VM-PSs is indicated in pink. For simplicity, three gene groups are separately depicted in three successive VM-PSs. See the text for details.

View larger version (118K): [in a new window]   Fig. 7. Roles of VM-bnl in tracheal development. vN and tN (N is an arbitrary integer) indicate VM-PS and Tr number, respectively. (A-C) Wild type. (D,E) bap-GAL4/+; UAS-bnl/+. (F,G) wgts embryo shifted up during early midstage 11 (5.36.3AEL). (H) twi-GAL4/+; UAS-dTCF-N/+. Late stage 11 to early stage 12 embryos are stained with FAS2, TIN or FAS3 as indicated. Lateral views are shown except for (B1, F1, H1), which are ventral views. Vertical bars, VM-PS borders. Panel interpretation is shown below. The region labeled with a bracket in A is enlarged in B2. (A-C) Relationship between VB budding and VM-bnl expression in wild type. (A) VB buds towards VM-bnl domains in a 1:1 fashion at late stage 11. (B) VB of Tr8 first touches the posterior vicinity of the TIN-expressing a region of VM-PS11. (C) At early stage 12, several cells touch with the p and a region expressing tin/bnl. (D,E) Misexpression of bnl in the entire VM caused misrouting (D) or bifurcation (E) of VB (arrows). (F-H) Reduction of tin/bnl expression domain using wgts mutant (F,G) or UAS-dTCF-N (H) causes misrouting of VB towards the p region of VM-PS10 (F2) or VM-PS10-11 border (H2), respectively. (I) VB shift in wg or WG signaling mutants is schematically shown.

View larger version (84K): [in a new window]   Fig. 5. Involvement of VM-hh in VM-PS subdivision. (A,B) Wild type. (C-D) hhts. (E,F) 48Y-GAL4/UAS-ciNZn. Late stage 11 (E) and stage 13 (A-D,F) embryos stained as indicated. (A-D) Elimination of hh activity during mid stage 11 to stage 12 (6.37.8 AEL) did not perturb the initiation of CON and VM-hh (data not shown), but caused loss of their expression at stage 13 (C,D). Note that CON expression in VM-PS3-5 still persists, even in the complete absence of hh activity (D; data not shown). (E,F) VM-hh expression was virtually normally initiated (E), but could not persist when UAS-ciNZn was driven by 48Y-GAL4 (F).

View larger version (96K): [in a new window]   Fig. 6. hh regulates gastric caecum development through dpp expression regulation. (A,D,F,G,I,J,L) Guts dissected at 25 AEL stained with phalloidin-FITC. Arrows indicate gastric caecum evaginations; asterisks indicate eliminated gastric caeca. Compare the gastric caecum size using the vertical broken line as a measure. (B,E,H,K) dpp RNA expression in stage 13 embryos with HH signaling defects. Large arrowheads indicate dpp expression in VM-PS3. dpp expression in VM-PS6,7 serves as an internal control (small arrowheads). (A-C) The wild-type embryo possesses four long evaginations of gastric caecum with similar size. In wild type, VM-PS3 dpp (red) expression almost overlapped that of VM-hh-lacZ (green) (inset in B). (C) The dorsal view of a wild-type stage 14 embryo stained for Dwnt4 RNA (red) and VM-hh-lacZ (green). Arrowheads indicate VM-PS3 gastric caecum primordia with hh expression. In hhts embryos shifted-up during mid-stage 11 to late stage 12 (6.3-9.3 AEL), gastric caeca were shortened or eliminated (D,F) and VM-PS3 dpp expression was significantly reduced in area and intensity (E). In embryos with 48Y-GAL4-driven UAS-ciNZn, gastric caecum was always short (G) and VM-PS3 dpp expression was reduced (H). (I) bap-GAL4/+; UAS-ciNZn/+ embryo. In hh13C (J) or twi-GAL4/+:UAS-ciNZn embryos (L), gastric caecum evagination was barely detectable. In these embryos, dpp expression in VM-PS3 was abolished (K; data not shown).

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