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

Ryan B. Green 2 Victor Hatini 1 Katherine A. Johansen 0 Xue-Jun Liu 0 3 Judith A. Lengyel ) 0 2 0 Department of Molecular, Cell and Developmental Biology, UCLA , Los Angeles, CA 90095-1606 , USA 1 Department of Cell and Developmental Biology, University of Pennsylvania, School of Medicine , 1223 BRB2, 421 Curie Boulevard, Philadelphia, PA 19104-6058 , USA 2 Molecular Biology Institute 3 Present address: The R. W. Johnson Pharmaceutical Research Institute , La Jolla, CA , USA Drumstick is a zinc finger protein that antagonizes Lines to control patterning SUMMARY Elongation of the Drosophila embryonic hindgut epithelium occurs by a process of oriented cell rearrangement requiring the genes drumstick (drm) and lines (lin). The elongating hindgut becomes subdivided into domains small intestine, large intestine and rectum each characterized by a specific pattern of gene expression dependent upon normal drm and lin function. We show that drm encodes an 81 amino acid (10 kDa) zinc finger protein that is a member of the Odd-skipped family. drm expression is localized to the developing midgut-hindgut junction and is required to establish the small intestine, while lin is broadly expressed throughout the gut primordium and represses small intestine fate. lin is epistatic to drm, suggesting a model in which localized expression of drm Cell rearrangement drives many morphogenetic processes. During vertebrate gastrulation, non-epithelial mesodermal cells intercalate and converge toward the midline in a process designated convergent extension, resulting in dramatic elongation of the embryonic axis (Warga and Kimmel, 1990; Keller et al., 1985). Rearrangement of non-epithelial cells also occurs during invertebrate development, including the formation of Drosophila ovarian terminal filaments and the migration of ovarian border cells (Godt and Laski, 1995; Montell, 1999). Cells can also rearrange while remaining constrained within an epithelial sheet. This type of rearrangement is responsible for elongation of both the Drosophila germ band and the C. elegans dorsal epidermis (Irvine and Wieschaus, 1994; Heid et al., 2001). Similarly, epithelial cell rearrangement causes elongation of developing tubes, including the sea urchin archenteron, C. elegans intestine, and Drosophila posterior spiracles (Ettensohn, 1985; Leung et al., 1999; Brown and Castelli-Gair Hombra, 2000). For a field of epithelial cells to change shape in a coherent and directed manner, cell rearrangement within the epithelium blocks lin activity, thereby allowing small intestine fate to be established. Further supporting this model, ectopic expression of Drm throughout the hindgut produces a lin phenotype. Biochemical and genetic data indicate that the first conserved zinc finger of Drm is essential for its function. We have thus defined a pathway in which a spatially localized zinc finger protein antagonizes a globally expressed protein, thereby leading to specification of a domain (the small intestine) necessary for oriented cell rearrangement. must be oriented. In the case of the elongating Drosophila germ band, this orientation depends on the patterning of the anteroposterior axis of the embryo (Irvine and Wieschaus, 1994). Later, however, during development of epithelial organs, cell rearrangement is oriented to newly established axes that are specific to the organ itself. Examples include the elongation of the insect Malpighian tubule relative to signals from the tip cell, epithelial migration during tracheal system development relative to localized FGF signals in surrounding mesoderm, and eversion of the leg imaginal disc in response to segmentally localized Notch signaling (Skaer, 1993; Bradley and Andrew, 2001; Rauskolb and Irvine, 1999). A central problem in epithelial morphogenesis, therefore, is to understand how the positional cues (i.e. spatial patterning) that orient cell rearrangement are established. The Drosophila hindgut provides a model system in which to investigate patterning and its role in orienting cell rearrangement. The embryonic hindgut is a single-layered epithelium that elongates by both cell rearrangement and cell shape change (Skaer, 1993; Lengyel and Liu, 1998; Iwaki et al., 2001). Genes controlling each step of the morphogenetic process (specification and internalization of the primordium, maintenance and elongation, and specification and patterning of subdomains) have been identified (Lengyel and Iwaki, 2002). In particular, the genes drumstick (drm) and lines (lin) are required for both patterning in the prospective small intestine and for the cell rearrangement that drives elongation of the hindgut epithelium. While drm is required to commit cells to the small intestine fate, lin represses this fate (Iwaki et al., 2001). By analysis of single and double mutants, we show here that drm and lin interact genetically, and that lin is epistatic to drm. The lin gene encodes a novel, globally expressed protein that is thought to be a transcriptional regulator (Hatini et al., 2000). We show here that drm encodes a small, zinc finger protein expressed in a dynamic, spatially localized pattern that is consistent with the drm mutant phenotype. Ectopic expression and biochemical interaction studies indicate that Drm antagonizes Lin activity, probably through direct binding. Thus, a relief-of-repression mechanism allows expression of genes that define the small intestine. The specification of this domain is essential for the oriented cell rearrangement that elongates the hindgut. MATERIALS AND METHODS Previously described mutant alleles used were: drm1 (Liu et al., 1999), lin2 (Hatini et al., 2000), Df(2L)tim02 (Myers et al., 1995), and Df(2L)ed1 (Reuter and Szidonya, 1983). The strong UAS-lin 8 (used here) and other weaker UAS-lin stocks (Hatini et al., 2000), the bynGAL4 hindgut-specific driver (Iwaki and Lengyel, 2002), and the 143fkh-GAL4 driver (Fuss and Hoch, 1998) have been previously described. The drmP1 lin2 double mutant was generated by recombination. UAS-lacZ is from the Bloomington Stock Center (Indiana University, Bloomington, IN). Generation of drm alleles Df(2L)drmP1 and Df(2L)drmP2 were generated by mobilization of the l(2)k10101 P element (Trk et al., 1993). New point mutations in drm were generated by ethyl methanesulfonate mutagenesis (Grigliatti, 1986). Briefly, mutagenized cn bw sp males were crossed to Gla/SM6b females; 17,757 F1 progeny chromosomes were tested for failure to complement either Df(2L)drmP1 or Df(2L)drmP2. To minimize the effects of second site mutations, newly isolated drm chromosomes were recombined with ast1 dppd-ho ed1 dpov1 cl1 and screened for cross-over events between ed (24D3) and dp (25A), thus replacing ~80% of the mutagenized chromosome. To identify altered nucleotides, new drm chromosomes were balanced over CyO-GFP (Casso et al., 2000) for selection and sequencing of homozygous genomic DNA. The three exons were amplified individually by (...truncated)