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doi: 10.1242/10.1242/dev.00548

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The T-box-encoding Dorsocross genes function in amnioserosa development and the patterning of the dorsolateral germ band downstream of Dpp

Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, New York NY 10029, USA

View larger version (103K): [in a new window]   Fig. 1. Genomic clustering, gene structure and phylogenetic analysis of the T-box genes Doc1, Doc2 and Doc3. (A) Arrangement of the three Dorsocross genes within a genomic region of about 40 kb. CG5194 is a predicted gene with no similarity to any known gene. The exon structures of Doc cDNAs are depicted below with the coding sequences hatched and the T-box domains in black. The exon-intron structure with the T-box spanning exons 2 to exons 5 is conserved among Doc1, Doc2 variant A and Doc3. (B) ClustalX-generated alignment of T-box domains from T-box genes of Drosophila melanogaster (Doc1, Doc2, Doc3, omb/optomotor-blind, H15, H15r/H15-related/CG6634, org-1/omb-related gene 1, byn/brachyenteron/trg) and human (marked Hs). Additional Tbx6/16-related members of the T-box family, which appear to form a separate subgroup, are included from zebrafish (Dr) and Xenopus laevis (Xl). The T-box core sequence was N- and C-terminally extended in order to include amino acids partially conserved between subfamily members. (C) Phylogenetic N-J tree derived from ClustalX analysis, based on the alignment shown in B and using 1000 bootstrap trials (bootstrap values at tree node represent confidence values; branches with values below 700 are generally considered as less reliable and below 500 as unreliable. Bar represents amino acid exchanges as a fraction of 1). Caenorhabditis elegans (Ce) Tbx9 was included as an outgroup member. GenBank Accession Numbers are, for Doc2A, AAM11544; for Doc2B, AAM11545; and for Doc3, AAM11543. A Doc1 protein sequence identical to ours has previously been submitted by R. Murakami and T. Hamaguchi (AB035412).

View larger version (116K): [in a new window]   Fig. 6. Loss of Dorsocross causes abnormal development and premature breakdown of the amnioserosa. Homozygous Df(3L)DocA mutant embryos (A,C,E,G) and heterozygous control embryos (B,D,F,H) were stained for amnioserosa markers as indicated. (A,B) Expression of hnt, as determined with anti-Hnt antibodies, initiates in the absence of Dorsocross (A), but fails to the reach wild-type levels especially in anterior areas of the amnioserosa by stage 9 (arrowheads; compare with B). Note comparable levels of Hnt in midgut primordia (amg, pmg) in A and B. (C-H) Anti-C15 antibody staining of stage 9 (C,D), late stage 12 (E,F) and stage 14 (G,H) embryos. C15 expression is not reduced in early DocA mutants (C) and large flattened nuclei are present in the amnioserosa similar to the wild-type situation (D). However, in DocA mutants there are some abnormally small nuclei in the amnioserosa (arrow) and germ band elongation is slightly aberrant. (E) C15 staining of a stage 12 DocA mutant embryo reveals a decrease in the size of most amnioserosa nuclei (arrow) and a broadening of the ectodermal C15 expression domain (arrowhead) when compared with F. Around stage 14, no large amnioserosa cells expressing C15 can be found in DocA mutants (G), there is a dorsal hole lacking C15 (arrowhead) and the germ band is not retracted. In the control embryo (H), C15 expression in the amnioserosa is still strong during this stage and in the dorsal ectoderm it shows a well-defined segmented pattern. DocA mutants were initially identified via absence of balancer-derived anti-ß-galactosidase staining. Various morphological features (reduction of dorsal head structures, incomplete germ band extension associated with an inwardly kinked posterior germ band, absence of germ band retraction, yolk displacement) were found to be consistently present in mutants, which allows reliable discrimination of mutant and control embryos without anti-ß-galactosidase staining after stage 8.

View larger version (89K): [in a new window]   Fig. 5. Mutagenesis of the Dorsocross locus and phenotypic rescue experiments. (A) Genomic map of the Dorsocross region showing known and predicted genes, deficiencies and P-insertions. Numbers indicate base pair coordinates of genomic sequences on chromosome 3L (BGPD release 3). Slashes indicate the omission of sequences owing to space limitations. Genes above the genomic line are transcribed from left to right and those below from right to left (introns are not displayed). EP(3)3556 was used for generating Df(3L)DocA and Df(3L)DocB, and EP(3)584 (male-sterile insertion in bol, located within the omitted sequence) for Df(3L)EP584MR2. Relevant breakpoints of previously known deficiencies were mapped by PCR from homozygous embryos. Dashes indicate uncertainty ranges of breakpoints. In situ hybridization confirmed the presence of mRNAs of all three Doc genes in Df(3L)Scf-R6, absence of Doc3 mRNA in Df(3L)Scf-R11, absence of Doc1 mRNA in Df(3L)29A6, and absence of all three Doc mRNAs in Df(3L)DocA and Df(3L)DocB homozygotes. Df(3L)DocA and Df(3L)DocB complement female sterility associated with smg1, whereas Df(3L)Scf-R6 and Df(3L)Scf-R11 do not. Therefore upstream smg sequences (hatched), including alternative smg start sites, are dispensable. The sequences at the breakpoints of Df(3L)DocA, Df(3L)DocB and Df(3L)EP584MR2 will be made accessible in FlyBase. (B-D) Mid stage 12 embryos stained with anti-Kr antibody. (B) Embryo homozygous for Df(3L)DocA (DocA mutant; composite of two focal planes), which lacks Kr in the amnioserosa (arrowheads). Kr in the nervous system serves as an internal staining control (white asterisks). (C) Doc1+2+3 RNAi embryo, which is a wild-type embryo injected with a mix of Doc1, Doc2 and Doc3 dsRNA (3'-fragments downstream of T-box) and shows a phenotype as with DocA mutants. (D) Control wild-type embryo injected with buffer only. (E) Stage 14 DocA mutant embryo showing absence of Kr staining in the amnioserosa region (arrowhead) and incomplete germ band retraction. Somatic muscle staining of Kr in E-G is denoted by black asterisks. (F) Stage 14 DocA mutant embryo with forced expression of Doc2 in the early amnioserosa (via c381-GAL4). Kr expression in the amnioserosa is rescued to a significant degree (arrow), as well as extended temporally. Retraction defects are fully rescued in this and the majority of other embryos. (G) Stage 14 DocA mutant embryo with forced expression of Doc3 as in F. There is some rescue of Kr expression in the amnioserosa (arrow) but little rescue of the germ band retraction defects. (H-J) Cuticle preparations of unhatched first instar larvae visualized by dark-field optics. (H) Df(3L)DocA mutants have a u-shaped phenotype owing to the failure of germ band retraction. A similar cuticle phenotype is observed in Doc1+2+3 RNAi embryos (I), but not in the wild-type control (J). as, amnioserosa; fk, filzkörper.

View larger version (101K): [in a new window]   Fig. 2. Embryonic expression pattern of Dorsocross. Nuclear Dorsocross proteins were detected by immunostaining with anti-Doc3+2 antibody (see Materials and Methods) and visualized either with DAB (brown in A-D,F-I,L) or fluorescent secondary antibodies (J). Dorsocross mRNA was detected by in situ hybridization with specific probes for Doc3 (E) or Doc1 (K). Images from fluorescent staining are combined ectodermal (E), subepidermal (J) or mesodermal (K) confocal sections. Views are lateral (B,D,E,G,I-K) or dorsolateral (C,F) with dorsal upwards and anterior towards the left, or dorsal with anterior towards the left (A,H,L). Embryos are oriented the same way in all figures and all non-flourescent images are taken using Nomarski optics. (A) Blastoderm stage embryo, (B) stage 7 embryo and (C) stage 8 embryo showing Dorsocross protein in the anlagen of the amnioserosa (as) and the procephalic neuroectoderm (pne). Amnioserosa nuclei are distinguishable by their larger size. (D) At stage 10, a metameric expression pattern (PS4, parasegment 4) has emerged in addition to continued expression in the amnioserosa. (E) Patches of Dorsocross expression (green) alternate with the tracheal placodes labeled by anti-Cf1a antibody staining (red) in the dorsolateral ectoderm of the embryonic trunk. (F,G) At stage 14, epidermal stripes are visible dorsally and ventrolaterally. (H) Enlarged view of stage15-16 embryo that has completed dorsal closure, showing expression in dorsally fused epidermal stripes (de) and in the dorsal pouch (dp), and two pairs of cardioblasts per segment (cb). (I) Stage 16 embryo focussed on Doc expression in Malpighian tubules (arrowhead). (J) Dorsocross expression in the pentascolopidial chordotonal organs (arrowheads, green), which are marked by their staining with mab 22C10 (red). Dorsocross-positive cells are identified as ligament cells based on their ventral juxtaposition to 22C10-labeled LCh5 neurons (arrowheads). Some epidermal Doc staining is also present in this image. (K) The metameric expression as seen in D includes the dorsal mesoderm, where Dorsocross-expressing cells (green) alternate with visceral mesoderm precursors that express bap (red). The combined sections include ectodermal expression seen in more radial areas [see broken line between mesoderm (ms) and ectoderm (ec)]. (L) Stage 11 embryo focussed on the bilateral caudal visceral mesoderm anlagen (cvm) close to the posterior tip of the germ band.

View larger version (88K): [in a new window]   Fig. 3. Dorsocross expression along the dorsal midline of blastoderm stage embryos requires dpp and zen. (A-D) Double-fluorescent staining for Doc3 RNA and nuclear Phospho-Mad (PMad) protein. An antibody specific for the activated (phosphorylated) form of the Dpp effector Mad allows monitoring Dpp activity. All views are dorsal, except the lateral view in D. The longitudinal stripe is absent from homozygous dppH46 embryos (B) when compared with the wild type (A). (C,D) Upon induction of ectopic Dpp via P{GAL4-nanos.NGT}40 and UAS-dpp, nuclear PMad and expression of Doc3 (as well as of Doc1 and Doc2, not shown) appear in a significantly broader longitudinal stripe when compared with A. PMad, but not Doc3, is also detected in ventral cells (D). (E) Double in situ hybridization for Doc3 mRNA (green) and zen mRNA (red) shows that the dorsal stripe of Doc expression appears during the time when refined zen expression is detected on top of the weaker broad zen pattern. (F) During mid stage 5, when zen transcripts in the broad dorsal domain have disappeared, and dorsal Doc3 and zen are expressed at peak levels, the widths of the Doc3 and zen domains are identical. (G) Dorsolateral view of heterozygous zen7/CyO embryo and (H) homozygous zen- mutant (zen7, also known as zenW36) stained with anti-Doc3+2 antibody. Very little Dorsocross protein is detectable along the dorsal AP axis in zen mutants. By contrast, expression is maintained in the head stripe and at the termini (foregut and hindgut primordia, more prominently at later stages). Embryos in A,B,G,H were analyzed at the beginning of gastrulation, when wild-type embryos have a fully formed dorsal stripe.

View larger version (106K): [in a new window]   Fig. 4. Combinatorial inputs from Dpp and Wg signaling regulate metameric Dorsocross expression in the dorsolateral ectoderm and mesoderm. (A,B) Doc3 in situ hybridization (green) and anti-PMad antibody staining (red) in wild-type stage 10 embryos seen from lateral (A) or ventral (B) views (only trunk regions are shown in this figure; v, ventral midline). Doc3 expression is activated within the nuclear PMad domain. (C) Ventral extension of the ectodermal PMad domain by ectopic Dpp leads to ventral extension of Doc3 patches. Ectopic Dpp was driven from a UAS-dpp transgene by ZKr-GAL4 in the ectodermal Krüppel domain (Frasch, 1995). (D,E) Wild-type stage 10-11 embryos double-stained with anti-Doc3+2 antibody (green) plus anti-En/Invected (red) (D) and anti-Doc3+2 antibody (green) plus anti-Wg (red) (E). Ectodermal Doc expression is centered around the Wg-stripes and overlaps partially with the En stripes (yellow ectodermal signals). (F) Homozygous wgCX4 mutant embryo stained as in E. Metameric Doc expression is absent from the ectoderm and mesoderm, although amnioserosa expression is not affected. All images are combined confocal sections within the specified germ layer. Doc-expressing cells flanking the amnioserosa (as) in D, and to lesser extent in E, are mesodermal.

View larger version (75K): [in a new window]   Fig. 7. Abnormal marker expression, cell cycle entry and premature apopotosis of amnioserosa cells in Doc mutant embryos. Shown are confocal fluorescent microscopic images with merged amnioserosa scans. (A-D) race in situ hybridization (green) and antibody staining (red) with anti-Hnt (A,B) and anti-C15 (C,D) at stage 12. race expression decreases in the amnioserosa of DocA mutants. Residual expression of race generally correlates with residual Hnt expression (arrow in A) and large C15-stained nuclei (arrow in C; arrowheads, small nuclei). In addition, there are increased levels of race mRNA in the posterior/dorsal head (asterisk) of DocA embryos. (E,F) Stage 10 embryos stained for race mRNA and phospho-Histone H3. Unlike in the wild-type (F), race-stained amnioserosa cells show nuclear phospho-Histone H3 staining in DocA mutants (E, arrow heads). (G,H) Stage 11 embryos after 30 minutes BrdU pulse labeling, double-stained with anti-BrdU (green) and anti-C15 antibodies (red) to visualize amnioserosa nuclei. Overlapping signals appear yellow. While normal amnioserosa cells are arrested in the G2 phase of cell cycle 14 and do not incorporate BrdU (H), BrdU incorporation is detected in a fraction of amnioserosa cells of DocA mutants (arrowheads in G). BrdU incorporation in dorsal ectodermal cells flanking the amnioserosa and other domains is seen in both mutant and wild-type embryos at this stage. (I,J) Stage 11 embryos stained for C15 (red), DNA (Hoechst, blue) and -tubulin (green) after fixation in the presence of taxol. Mitotic spindles in the amnioserosa of DocA mutants (I, arrowheads) indicate dividing amnioserosa cells, which are not seen in wild-type embryos (J). (K,L) Detection of apoptotic cell death in late stage 12 embryos by TUNEL is shown in green and staining with anti-C15 is shown in red. At this stage, there is significant apoptosis within the amnioserosa in DocA mutants (K, arrowheads), but none in wild type (L), although apoptosis can be detected in the head and other regions of wild-type embryos. Most apoptotic amnioserosa cells have already lost C15 expression, but are clearly localized within the amniosera layer. Mutant embryos were identified by triple-staining with anti-ß-galactosidase (A-D; not shown) or by the absence of anti-Doc2 staining performed in parallel to anti-C15 staining (G,I,K). G,H are at 1.75x greater magnification than A-F,K,L; I,J are at 2.5x greater magnification than A-F,K,L.

View larger version (54K): [in a new window]   Fig. 8. Dorsocross regulates the patterning of the dorsolateral ectoderm. (A) Fluorescent double-staining with anti-Doc3+2 antibodies (green) and anti-Wg (red). After an initial overlap of expression (see Fig. 4E), at stage 11 there is complementary expression of Doc and wg as the wg stripes become interrupted in the dorsolateral ectoderm. (B,C) Anti-Wg staining (brown; blue anti-ß-galactosidase staining indicates TM3, eve-lacZ-balanced control embryos) of stage 11 embryos. Note persistence of continuous stripes in DocA mutants (B, arrowhead), which contrasts with interrupted stripes in control embryos (C, arrowheads). (D) Expression of Dorsocross (anti-Doc3+2, green) and ladybird (anti-Lbe, red) in the embryonic trunk at stage 11/12. Doc patches are observed between Lb patches similar to the spatial arrangement seen in A. (E,F) Anti-Lbe staining (plus anti-ß-galactosidase staining, blue) of stage 14 embryos. (E) Continuous lb stripes are visible in the ectoderm of DocA mutants. (F) Normal lb expression pattern with separated dorsal patches and ventral stripes in the ectoderm in control embryos. Small groups of cells expressing lb in the lateral region correspond to muscle precursors.

View larger version (95K): [in a new window]   Fig. 9. Ectopic expression of Dorsocross represses wingless. (A) wg expression in stage 11 control embryo (UAS-Doc2/CyO). (B) Embryo of similar stage expressing Doc2 ectopically in the whole ectoderm (e22c-GAL4/UAS-Doc2). Ventral wg expression is missing (see arrowhead) and dorsal wg expression is almost continuous, although with reduced levels. (C,D) Dorsocross (red) and wg (green) expression in imaginal wing discs from 3rd instar larvae detected by flourescent double-staining using anti-Doc2+3 and anti-Wg antibodies. Dorsal is upwards and anterior towards the left. (C) Wild-type wing disc. (D) Wing discs ectopically expressing Doc2 in the dpp expression domain (UAS-Doc2/+;dpp.blk1-GAL4/+). Note the interruption of wg expression at the intersection of ectopic Doc2 and Wg stripes (arrowheads). (E,F) Leg disc of 3rd instar stained as in C and D. (E) Wild-type leg disc with wg expression in anterior/ventral territories. Endogenous Doc protein in dorsal proximal areas of the disc is detected at low levels (red arrowheads). (F) Leg disc from UAS-Doc2/+;dpp.blk1-GAL4/+ larvae. wg expression is repressed by ectopic Doc2 in the central area that normally produces distal parts of the leg (arrowheads). (G) Wild-type wing. (H) Wing derived from a disc with a genotype as in D, which lacks distal structures (arrowheads). Wing veins appear broadened as well. (I) Adult male fly showing a phenotype of intermediate strength caused by ectopic dpp.blk1-driven Doc2. Arrowheads indicate aristaless antennae and shortened legs.

View larger version (21K): [in a new window]   Fig. 10. Summary of currently known regulatory networks during amnioserosa development. Dorsocross genes act downstream of Dpp and zen, and upstream of hnt and Kr. Other pathways, which include C15, act in parallel with Dorsocross (see Discussion).