QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online March 4, 2005
doi: 10.1242/10.1242/dev.01687

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, H.-H. Articles by Frasch, M. Search for Related Content PubMed PubMed Citation Articles by Lee, H.-H. Articles by Frasch, M.

Nuclear integration of positive Dpp signals, antagonistic Wg inputs and mesodermal competence factors during Drosophila visceral mesoderm induction

Hsiu-Hsiang Lee^* and Manfred Frasch

Brookdale Department of Molecular, Cell and Developmental Biology, Box 1026, Mount Sinai School of Medicine, New York, NY 10029, USA

View larger version (52K): [in a new window]   Fig. 1. Summary of signaling and transcriptional pathways during the induction of trunk visceral mesoderm. Shown are three parasegments of the mesoderm (divided into P and A domains) (Azpiazu et al., 1996) of a stage 10 embryo stained for bap mRNA (purple) and Eve protein (brown). The expression domains of Tin (schematically shown in blue) and the relevant Slp domains (red) are within the mesoderm, whereas Dpp (yellow) and Wg (brown) are secreted from the overlying ectoderm. Inductive signals are represented by hatched arrows and transcriptional interactions are represented by solid arrows.

View larger version (62K): [in a new window]   Fig. 5. Evolutionary conservation of bap enhancer sequences and binding motifs. Shown are alignments of enhancer sequences of bap3.2 (D. melanogaster), bapV2 (D. virilis) and corresponding genomic sequences from D. yakuba (D. yak.), D. pseudoobscura (D. pse.) and D. ananassae (D. ana.). Colored boxes above the sequences with unbroken lines indicate the extent of DNAseI footprints on D. melanogaster sequences, boxes with black broken lines delineate highly conserved DNA stretches (C1 and C2), and colored boxes within the sequences denote core binding motifs for the respective binding factors. Nucleotides altered by in vitro mutagenesis for in vivo testing of binding site activities are shown on top of the D. melanogaster sequence (for Slp/Bin site mutations, see Fig. 7). For R1-R3 motifs, see text and Fig. 8. The R1 sequence is not readily detectable in the other species but the 5' region of bap3 (not shown) contains additional R-related motifs that are conserved and may have functionally redundant activities.

View larger version (71K): [in a new window]   Fig. 7. Functional dissection of the Slp binding sites in the bap enhancer. (A) Wild-type and mutated sequences within the region protected by Slp. The inverted repeat of canonical forkhead domain-binding motifs is in black boxes and the CAAA type of Slp-binding motifs are underlined in red. Unaltered sequences are represented by dashes below, and deleted sequences are indicated as a bracketed unbroken line. (B) Activity of the parental bap3.2.1-lacZ construct used as a control. (C) bap3.2.1-slp-m1-lacZ is not active in the mesoderm, while in the dorsal ectoderm it is active in metameric domains and there is weak ectopic activity between these domains. (D) bap3.2.1-slp-m2-lacZ shows very weak activity in the mesoderm and similar ectodermal activity as with bap3.2.1-slp-m1-lacZ. (E) bap3.2.1-slp-m3-lacZ shows weakened activity in the mesoderm and similar ectodermal activity as with bap3.2.1-lacZ. (F) bap3.2.1-slp-m4-lacZ activity is similar to that of the parental bap.3.2.1-lacZ (mesodermal clusters have physically merged at this slightly later stage). (G) bap3.2.1-slp-m5-lacZ shows lack of mesodermal activity and largely uniform dorsal ectodermal activity along the anteroposterior axis. (H,I) Fluorescent double staining for Slp (red) and ßGal (green) in stage 10 embryos. bap3.2.1-lacZ expression (H) is complementary to that of Slp, whereas bap3.2.1-slp-d1-lacZ expression (I) overlaps with Slp.

View larger version (72K): [in a new window]   Fig. 8. Sequences required for preventing the induction of bap enhancer activity in the dorsal ectoderm. (A) Summary of tested enhancer derivatives and their activities in the dorsal mesoderm (ms) and ectoderm (ec). R1, R2 and R3 denote native motifs with putative repressing activities, whereas tinD1a and tinD1b denote related sequence motifs from the tinD enhancer of tinman. (B) Sequence alignments of motifs thought to confer ectodermal repression from the bap3.2 and tinD enhancers. (C-H) Dorsal views of early stage 11 ßGal-stained embryos carrying various reporter constructs (arrow, mesoderm; arrowhead, ectoderm). (C) bap3.2-lacZ shows almost complete repression in the dorsal ectoderm. (D) bap3.2.1-lacZ shows complete de-repression in the dorsal ectoderm. (E) bap3.2R3-lacZ shows low levels of de-repression in the dorsal ectoderm (small arrow; see comment in Fig. 6 legend regarding large ectodermal cells). (F) bap3.2R1-2-lacZ and (G) bap3.2R1-3mut-lacZ show strong de-repression of enhancer activities in the dorsal ectoderm. (H) The addition of tinD1 sequences to bap3.2.1 prevents ectopic induction in the ectoderm.

View larger version (89K): [in a new window]   Fig. 2. Conserved bap expression and activities of bap enhancers from D. melanogaster and D. virilis. (A-H) Lateral views, (I-L) dorsal views, (M-O) cross-sections and (P) ventral view. (I-P) Arrows indicate the dorsal mesoderm and arrowheads indicate the dorsal ectoderm. (A) Stage 10 D. melanogaster embryo hybridized with a D. melanogaster bap probe, which detects bap mRNA expression in the trunk visceral mesoderm (TVM) and hindgut visceral mesoderm (HVM) primordia (foregut/FVM expression out of focus). (B) Hybridization of a stage 10 D. virilis embryo with a D. virilis bap probe shows an identical expression pattern as in D. melanogaster. (C) D. melanogaster bapDS3.5-lacZ (stage 10) recapitulating bap expression in HVM and FVM. Low levels of TVM expression are also seen. (D) D. virilis bapDS4.6-R-lacZ activity is nearly identical to that of D. melanogaster bapDS3.5-lacZ. (E,F) Stage 14 embryos carrying the same constructs as embryos in C,D, respectively, show foregut and hindgut visceral mesoderm expression. (G) D. melanogaster bap3-lacZ recapitulates the TVM pattern of bap mRNA expression during stage 10. (H) D. virilis bapDS2.7-R-lacZ activity is similar to D. melanogaster bap3-lacZ activity. (I) D. melanogaster bap3.2-lacZ activity in stage 11 embryo is largely confined to the TVM primordia, although there are traces of ectopic activity in the dorsal ectoderm (arrowhead). (J) D. virilis bapV1-lacZ activity is similar to D. melanogaster bap3.2-lacZ activity. (K) D. melanogaster bap3.2.1-lacZ embryo (stage11) showing ectopic segmented enhancer activity in the dorsal ectoderm in addition to normal mesodermal expression. (L) D. virilis bapV2-lacZ showing ectopic ectodermal and largely normal mesodermal enhancer activity, similar to D. melanogaster bap3.2.1. (M) Cross-sectioned bap3-lacZ embryo (stage 10) showing exclusive dorsal-mesodermal enhancer activity. (N) bap3.2-lacZ with largely mesodermal expression but weak ectopic ectodermal expression. (O) bap3.2.1-lacZ showing equally strong activity in dorsal mesoderm and dorsal ectoderm. (P) Stage 9 embryo stained for bap mRNA (green) and phospho-Mad (red), showing coincidence of the ventral borders of nuclear pMad and bap mRNA expression.

View larger version (22K): [in a new window]   Fig. 3. bap enhancer regions and reporter constructs from D. melanogaster (A) and D. virilis (B). A restriction map of the genomic locus including the bagpipe (bap) transcription unit (5' to the left) is shown at the top of each panel. Shown below are the genomic fragments tested in reporter constructs, with the arrowhead indicating their orientations in the construct (arrowheads point towards basal promoter). bapH2-1.2 (D. melanogaster), bapDS2.7-R (D. virilis) and their respective subfragments are shown at higher magnification. The in vivo reporter gene expression patterns are indicated on the right-hand side. Identified enhancer regions are summarized at the bottom of each panel. B, BamHI; R, EcoRI; H, HincII; S, SalI; ec, ectoderm; ms, mesoderm; FVM, foregut visceral mesoderm; HVM, hindgut visceral mesoderm; TVM, trunk visceral mesoderm.

View larger version (74K): [in a new window]   Fig. 4. DNAseI protection experiments with candidate transacting factors on bap3.2.1 enhancer DNA. 32P-labelled probe was tested with two different amounts (1x and 3x, see Materials and methods) of bacterially expressed GST-fusion proteins of Bap, Tin, Slp, Mad, Medea and Bin, as well as BSA as a control. C+T sequencing ladder is shown on the left of each blot and a schematic drawing of protected regions on the right.

View larger version (73K): [in a new window]   Fig. 6. In vivo requirements for binding sites of Smad, Tin and Bap proteins, and for other conserved sequences. (A) Schematic representations of bap3.2.1 and its mutated derivatives with a summary of their in vivo activities (ms, dorsal mesoderm; ec, dorsal ectoderm). (B-I) Dorsal views of stage 11 embryos. Arrow indicates mesodermal layer and arrowhead indicates ectodermal layer. (B) Activity of the parental bap3.2.1-lacZ construct. (C) Mutations in the Bap-binding site cause a slight and transient reduction of mesodermal enhancer activity. (D) Mutations in the Tin-binding site cause a loss of enhancer activity in the mesoderm. (E) Mutations in the Mad/Medea-binding site 1 cause a loss of enhancer activity in both ectoderm and mesoderm. (F) Mutations in the Mad/Medea-binding site 2 nearly abolish enhancer activity in both ectoderm and mesoderm. (G) Deletion of DNA sequences containing the Mad/Medea-binding sites 3 and 4 causes a strong reduction of ectodermal and mesodermal enhancer activity. (H,I) Mutations within the conserved sequence C1 of the enhancer from D. melanogaster (H) and D. virilis (I) cause a loss of enhancer activity in both ectoderm and mesoderm. (The observed expression within single ectodermally derived cells in each hemisegment is an artificial effect from the transformation vector.)

View larger version (82K): [in a new window]   Fig. 9. Molecular switching of Dpp-responsive enhancer activities in the dorsal mesoderm. The schematic diagram summarizes the molecular basis for the reciprocal activities of the bap and eve enhancers in the A and P domains of the dorsal mesoderm. In the A domains, the differential activities are due to the Wg-dependent relief of dTCF-associated co-repressors at the eve enhancer and to the Wg-dependent loading of Slp/co-repressor complexes at the bap enhancer. Conversely, in the P domains where Wg signaling is absent slp is not induced, which allows the bap enhancer to be active, while dTCF/co-repressor complexes keep the eve enhancer `off'. Owing to the lack of Wg-responsive sequences, the tin enhancer is `on' in both domains of the dorsal mesoderm. For simplicity, only one site of each type is depicted and various sites binding yet unknown factors involved in activation or ectodermal repression are not depicted.