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Drumstick is a zinc finger protein that antagonizes Lines to control patterning and morphogenesis of the Drosophila hindgut

Ryan B. Green¹, Victor Hatini², Katherine A. Johansen³, Xue-Jun Liu^3,* and Judith A. Lengyel^1,3,

¹ Molecular Biology Institute,
³ Department of Molecular, Cell and Developmental Biology, UCLA, Los Angeles, CA 90095-1606, USA
² Department of Cell and Developmental Biology, University of Pennsylvania, School of Medicine, 1223 BRB2, 421 Curie Boulevard, Philadelphia, PA 19104-6058, USA
^* Present address: The R. W. Johnson Pharmaceutical Research Institute, La Jolla, CA, USA

View larger version (122K): [in a new window]   Fig. 1. lin is epistatic to drm. Whole-mount embryos were stained with anti-Crb (A-H,U-X) or anti-En (I-L), hybridized in situ with probes for upd (M-P) or hh (Q-T), or fixed and sectioned transversely (E-H). The wild-type hindgut (A) consists of three morphologically distinct domains: small intestine (SI), large intestine (LI), and rectum (RE). Boundary cell (BC) rings, labeled strongly with anti-Crb, separate the SI and LI anteriorly and the LI and RE posteriorly. Two boundary cell rows also run the length of the LI dividing it dorsoventrally, as seen in transverse section (two arrows in E). In drm embryos (B), the SI is missing, the cells of the LI are columnar, and the BC rows are duplicated (four arrows in F). In both lin (C,G) and drm lin (D,H) embryos, the LI is missing, the cells are cuboidal, and the BC rows are absent. In wild-type embryos, En is expressed in the dorsal portion of the LI (I, black arrowhead). In drm embryos (J), En is expressed throughout dorsal hindgut (black arrowhead), but is not expressed in the rectum. In lin (K) and drm lin (L) embryos, En-expressing cells are not present (white arrowhead). In wild-type embryos (M, arrowhead), upd is expressed within the SI. In drm embryos (N), upd is not expressed (white arrowhead). In lin (O) and drm lin (P) embryos, expression of upd is expanded throughout most of the hindgut (wide arrowhead), but not the RE. In wild-type embryos, hh is expressed in both the SI and RE (Q, arrowheads). In drm embryos (R), hh is expressed in the RE, the ventral portion of the LI, and at the junction between the hindgut and midgut (arrowheads). In lin (S) and drm lin (T) embryos, expression of hh is expanded throughout most of the hindgut (wide arrowhead), but not the RE. In Crb-stained wild-type embryos (U), the proventriculus (PV) forms a multi-layered valve-like structure between the esophagus (ES) and anterior midgut (arrows in U-X indicate junction with anterior midgut). In drm embryos (V), the PV does not fold properly. In both lin (W) and drm lin (X) embryos, the entire foregut appears shorter and wider, and the PV does not form (arrows).

View larger version (37K): [in a new window]   Fig. 2. Molecular characterization of drm. (A) Physical map between male-specific lethal-2 (msl-2) and foraging (for) (cytological regions 23F3 to 24A3). Genomic DNA in P1 phage clones DS01379 and DS01340 is shown above the map, with the 18.1 kb fragment used for genomic rescue experiments shaded in gray. Characterized genes are shown with arrows representing their directions of transcription, as annotated in GadFly (Rubin et al., 2000b). The l(2)k10101 P element, mobilized to generate deficiency alleles, is represented by an inverted triangle. Below, deficiencies are represented by open bars with breakpoint uncertainties shown in gray. Df(2L)tim02 and Df(2L)ed1 were used for rough mapping of the drm1 allele. The breakpoints of Df(2L)drmP1 and Df(2L)drmP2 were mapped molecularly. B, BamHI. (B) Expanded view of the 11.9 kb BamHI fragment that includes the drm gene. The I element in the drm1 chromosome is inserted 35 bp upstream of the transcription start site. The drm transcription unit is 8.5 kb with three exons spliced together to form a 2.5 kb mRNA. An alternate first exon (1') was found in 10% of 5' RACE products, and several alternate transcriptional stop sites (dotted lines in exon 3) were present in 3' RACE products. The drm ORF (black) spans the second splice junction. (C) Northern blot of 50 µg total embryonic RNA (0-17 hours) and 1 µg poly(A) RNA shows the approximately 2.5 kb drm mRNA. (D) Schematic representation of the Drm protein folded to form one C2H2 and one C2HC zinc finger. Residues conserved in the canonical C2H2 zinc finger are shaded gray and residues altered in the drm point mutants are black (see Table 1).

View larger version (50K): [in a new window]   Fig. 3. Drumstick is a member of the Odd-skipped family of zinc finger proteins. Cytological map showing the proximity of drm, sob, odd and bowl (A). Drm, Odd, Sob and Bowl are similar in their zinc finger domains, but otherwise share little sequence similarity; protein lengths are indicated to the right (B). Alignment of the zinc fingers from Drm, Odd, Sob, and Bowl (C). The zinc fingers from Mus musculus mOsr1 and mOsr2 (and its alternatively spliced form, mOsr2[alt]), are shown for comparison (mouse and human Osr proteins are identical within the zinc finger motifs). Identical and similar residues are shaded black and gray, respectively (BOXSHADE, v. 3.2). Dashes indicate gaps in the alignment, and dots indicate amino acid residues (not shown) outside the zinc finger domains. Conserved residues in the canonical C2H2 zinc finger are shown below. Asterisks above the Drm sequence denote single residues altered in drm mutants (see Table 1 and Fig. 2D), and the diamond denotes the second exon splice donor site where the drm3 roo element is inserted.

View larger version (199K): [in a new window]   Fig. 4. Expression of drm during embryogenesis. Whole-mount in situ hybridization with drm-specific probe shows the dynamic expression pattern of drm between embryonic stages 5 and 13 (A-H). drm expression is first observed at stage 5 in a ventral crescent in the posterior midgut-hindgut primordium (at 10% EL), a weakly stained anterior spot in the foregut-proventriculus primordium (at 95-100% EL) (black and white arrows in A-H indicate expression in posterior and anterior gut, respectively), and in seven transverse stripes (arrowheads; A). By stage 6, the posterior crescent has expanded to encircle the amnioproctodeal plate (B, dorsolateral view), and secondary transverse stripes arise between the initial seven to give a total of 14. Between stages 7 and 9, drm-expressing cells internalized with the proctodeal invagination are located at the midgut-hindgut junction (C-E). At stage 10, drm is expressed in the invaginating stomodeum and transiently in the Malpighian tubule buds (F). Between stages 11 and 13, drm expression is refined to the posterior midgut, the ureters of the Malpighian tubules, the anterior small intestine, the proventriculus anlage, and at the anterior boundary of each segment. Expression is also observed in the pharynx and stigmatophore. Whole-mount drm-GAL4:UAS-lacZ embryos stained with anti-ß-gal (I-K) reveal, by stage 13, lacZ expression in more extensive domains (both anteriorly and posteriorly) than those seen for drm mRNA expression at the same stage (I, dorsal view; compare Fig. 4H). At stage 16 (J and K, higher magnification), ß-gal is present throughout the entire proventriculus, anterior midgut, posterior midgut, Malpighian tubules, and small intestine. Dotted lines outline the proventriculus (J), posterior midgut, small intestine, and large intestine (K). MTB, Malpighian tubule buds; MT, Malpighian tubules; PV, proventriculus; AMG, anterior midgut; PMG, posterior midgut; HG, hindgut; SI, small intestine; LI, large intestine.

View larger version (75K): [in a new window]   Fig. 5. Drm and Lin antagonize each other in vivo. Whole-mount embryos were stained with anti-Crb (A-D) or anti-En (E,F), hybridized in situ with probe for upd (G,H) or hh (I,J), or fixed and sectioned transversely (G,H). Ectopic expression of Drm throughout the hindgut with the byn-GAL4 driver produces a lin-like phenotype as observed by morphology of the hindgut (A; compare Fig. 1C), by hindgut epithelial cell shape and absence of boundary cell rows (C; compare Fig. 1G), by the lack of expression of En in the large intestine (E, white arrowhead; compare Fig. 1K), and by the posterior expansion of upd and hh from the small intestine (G and I, wide arrowheads; compare Fig. 1O and 1S). Ectopic expression of Lin throughout the hindgut using the byn-GAL4 driver produces a drm-like phenotype as observed by the morphology of the hindgut (B; compare Fig. 1B), hindgut epithelial cell shape and duplication of boundary cell rows (D, four arrows, compare with Fig. 1F), the expression of En in the dorsal large intestine (F, black arrowhead; compare Fig. 1J), the absence of upd expression in the small intestine (H, white arrowhead; compare Fig. 1N), and the expression pattern of hh (J, black arrowheads; compare Fig. 1R).

View larger version (70K): [in a new window]   Fig. 6. Structure-function analysis of Drm-Lin interaction in vitro and in vivo. (A) Drm constructs. The full-length, wild-type Drm construct contains two zinc fingers (ZF1 and ZF2). The N-Drm construct consists of the 25-residue N-terminal portion of Drm, and the C-Drm construct contains the C-terminal 63-residue domain, including the two zinc fingers. R46C is the strong drm6 missense mutation within ZF1, and C57G is a missense mutation in one of the conserved zinc-binding Cys residues of ZF2. The ability of each derivative to bind Lin in vitro was determined by coimmunoprecipitation of epitope-tagged Drm and Lin from Schneider S2 cells (B). Lin binds with high affinity to full-length Drm and C-Drm, with lower affinity to C57G and C-Drm(C57G), and does not bind to N-Drm, R46C, or C-Drm(R46C). Control samples of cell lysates show that Lin was expressed in all transfection assays. To assess the effect of these constructs in vivo, byn-GAL4 was used to drive expression of each throughout the hindgut, and the phenotype characterized by anti-Crb staining (C). Expression of N-Drm results in a wild-type appearing hindgut, while expression of C-Drm produces a lin-like phenotype similar to the gain-of-function phenotype produced by ectopic expression of full-length Drm (compare Fig. 5A). Expression of R46C results in a wild-type hindgut while C57G produces a lin-like phenotype. +++ or +, strong or weak interaction, respectively; –, no interaction.

View larger version (13K): [in a new window]   Fig. 7. Model for small intestine specification by Drm-mediated relief of Lin repression. Lin is expressed broadly throughout the hindgut primordium and represses small intestine fate. Drm expression is localized to the anterior hindgut primordium where it represses Lin activity, thereby allowing the small intestine to be specified. See text for details.