u-shaped encodes a zinc finger protein that regulates the proneural genes achaete and scute during the formation of bristles in Drosophila

Abstract

The pattern of the large sensory bristles on the notum of Drosophila arises as a consequence of the expression of the achaete and scute genes. The gene u-shaped encodes a novel zinc finger that acts as a transregulator of achaete and scute in the dorsal region of the notum. Viable hypomorphic u-shaped mutants display additional dorsocentral and scutellar bristles that result from overexpression of achaete and scute. In contrast, overexpression of u-shaped causes a loss of achaete-scute expression and consequently a loss of dorsal bristles. The effects on the dorsocentral bristles appear to be mediated through the enhancer sequences that regulate achaete and scute at this site. The effects of u-shaped mutants are similar to those of a class of dominant alleles of the gene pannier with which they display allele-specific interactions, suggesting that the products of both genes cooperate in the regulation of achaete and scute. A study of the sites at which the dorsocentral bristles arise in mosaic u-shaped nota, suggests that the levels of the u-shaped protein are crucial for the precise positioning of the precursors of these bristles.

Figure 1

Schematic representation of the bristle patterns on the head and thorax of different mutant combinations of ush. The wild-type pattern is shown in a. In ush mutants, the dorsocentral bristles (DC) and the scutellar bristles (SC) of the thorax and the postvertical bristle (PV) on the head are the most frequently affected. The rest of the bristle pattern is normal. For each genotype, at least 30 hemithoraces were examined. Bristles present > 50% of the time are represented as black dots (wild-type positions) and blue circles (ectopic bristles), those present 10%–50% of the time are represented as yellow circles, and those present <10% of the time as red dots. (a) ANP and PNP are anterior and posterior notopleural bristles, respectively. (b–d) Three viable combinations forming an allelic series for the number of DC and SC bristles of the thorax and the PV on the head (see text). ush^rev18 is a deficiency uncovering several genes, including ush, and will be called ush⁻. An allele specific interaction exists between ush and pnr^D mutants (e–g). pnr^D/+ flies (f) display additional bristles (DC and SC) and loss of PV bristles (Ramain et al. 1993), a phenotype that is suppressed by three doses of ush⁺ (e) or enhanced when only one copy of ush⁺ (g) is present in these flies. The thoracic territories affected by the lack of ush function as revealed by clonal analysis (see text) are presented in h. The pink area denotes a territory where ush is required for cell viability and the green area denotes a territory where ush is required for the normal positioning of the DC bristles, part of this territory is also required for the fusion of the dorsal midline. When both ac and sc are nonfunctional, as in ac³ sc^10-1 flies, no bristles form whether or not ush is present (j and i).

Figure 2

The notal bristle phenotypes seen after a loss or an excess of ush expression. In the dorsocentral area of wild-type animals, two DC macrochaetes form in each hemithorax (A), whereas ush hypomorphic loss-of-function mutants display an excess of DC macrochaetes (ush^rev24/ush⁻ is shown in B). In contrast, overexpression of ush leads to loss of the DC bristles (hs–ush is shown in C). Absence of ush function also affects the formation of DC bristles, as revealed in clones of cells mutant for null alleles (D–F). Clones of mutant cells were generated during the larval stages (see Materials and Methods for details and complete genotypes). The mosaic border of the clones is indicated by a black line (D–F). A clone mutant for ush^TgR + 1 (D) shows the formation of wild-type DC bristles that are displaced from the normal site and have formed at the borderline separating wild-type and mutant cells. In large clones encompassing the entire dorsocentral region, no DC bristles form (E). However, ush function is not required for the construction of bristles in this area because occasionally an additional mutant DC bristle develops (arrow in F). (G–I) Drawings of clones situated in the dorso-central area. (○) Bristles of the wild-type genotype that have formed at ectopic positions at the borders of the mutant clones; (•) the normal positions at which these bristles should have been situated.

Figure 3

Analysis of the formation of bristle precursors in ush mutants and after overexpression of ush, and the spatial expression of ush transcripts. Expression of ac and sc was followed by using the enhancer trap lines ac–lacZ (A–C) and sca–lacZ (E–H), which are expressed in the proneural clusters (see text). The position of the macrochaete precursors was studied with the enhancer trap line marker A101 (D,I,J,K,L). Wing discs were dissected from late third-instar mutant and wild-type larvae. Wild-type discs are shown in A, E, and I. Arrowheads in A–C and E–G point to the DC cluster detected by both enhancer trap lines, and arrows point to the DC precursor cells in D and I–L. Many more cells express ac–lacZ (B) and sca–lacZ (F) around the dorsocentral site in ush^rev24/ush^rev18, and staining extends into the scutellar region where more scutellar bristles develop in this genotype. A cluster of DC precursors arises in ush^SW42/ush^TgR + 1 mutant discs (arrow in J). In contrast, when ush is overexpressed using either a hs–ush (C,G,K) or UAS–ush driven by the GAL4 line pnr^MD237 (H,L), the reduction of ac–lacZ (C) and the loss of expression of sca–lacZ (G) at the dorsocentral site indicate a severe reduction of ac–sc expression in the DC proneural cluster. No A101-positive cells (arrow) are seen at the dorsocentral site in hs–ush (K) and pnr^MD237/UAS–ush (L) discs. However, in hs–ush flies, the scutellar bristles develop (see Fig. 2C) and A101 lacZ staining is seen in the bristle precursors (K), although they fail to appear in the pnr^MD237/UAS–ush animals (L). (D) The in situ expression pattern of ush in the wing disc as revealed by digoxygenin-labeled probes. Expression is strong at the dorsal side including the scutellar territory and fades rapidly at the border of the area from which the dorsocentral bristles arise. Expression is also detected in the hinge region and in the presumptive region of the posterior pleura. The arrow points to dorsocentral precursors simultaneously labeled with A101.

Figure 4

ush regulates ac and sc expression through a dorsocentral-specific enhancer element. The dorsocentral specific enhancer element drives lacZ expression in the dorsocentral region of the thoracic discs (Gomez-Skarmeta et al. 1995). lacZ staining is shown in thoracic discs with both the DC–enhancer–sc–lacZ (A–C) and the DC–enhancer–ac–lacZ (D–F). Compared with wild-type discs (A,D), lacZ expression detected in ush^SW42/ush^TgR + 1 mutant wing discs (B,E) in the dorsocentral area is stronger and covers a larger domain, whereas it is almost absent when ush is overexpressed (pnr^MD237/UAS–ush) (C,F).

Figure 5

Structure and protein sequence of the gene ush. (A) Genomic map of the ush region. Proximal is to the right. The localization of some mutant ush alleles corresponding to deletions (ush^rev24, ush^rev18), to an insertion of a TE element (TE99) and to an insertion of a P element (ush¹⁵¹³) or to an inversion [In(2L)Tg^R + 1] is shown. Solid bars denote the direction of the deletions and incertitude concerning the precise breakpoint is indicated by hatched boxes. The breakpoint associated with In(2L)Tg^R + 1 is localized between +24 and +28 kb on the map and is symbolized by a hatched box. The transposon insertion site of ush¹⁵¹³ is shown by an arrow (0 kb on the map). The insertion point of the TE element is localized in the region designed by a hatched box (between −2 and 0 kb on the map). The intron/exon structure seen by comparison between the cDNA and the genomic DNA is indicated. The genomic region included in the cosmid cos4 and located between +2 and +35 kb on the map, allows almost complete rescue of ush null alleles. The restriction sites shown are EcoRI, BamHI, HindIII, NotI, which correspond to E, B, H, and N, respectively. (B) The sequence of the Ush protein corresponding to 1191 amino acids was predicted from both genomic and cDNA pU4.3 clones. The acidic region located in the amino-terminal part of Ush is boxed. Putative zinc fingers of both CCHC and CCHH type motifs are shown in grey, the CCHC ones are also boxed.

Figure 5

Structure and protein sequence of the gene ush. (A) Genomic map of the ush region. Proximal is to the right. The localization of some mutant ush alleles corresponding to deletions (ush^rev24, ush^rev18), to an insertion of a TE element (TE99) and to an insertion of a P element (ush¹⁵¹³) or to an inversion [In(2L)Tg^R + 1] is shown. Solid bars denote the direction of the deletions and incertitude concerning the precise breakpoint is indicated by hatched boxes. The breakpoint associated with In(2L)Tg^R + 1 is localized between +24 and +28 kb on the map and is symbolized by a hatched box. The transposon insertion site of ush¹⁵¹³ is shown by an arrow (0 kb on the map). The insertion point of the TE element is localized in the region designed by a hatched box (between −2 and 0 kb on the map). The intron/exon structure seen by comparison between the cDNA and the genomic DNA is indicated. The genomic region included in the cosmid cos4 and located between +2 and +35 kb on the map, allows almost complete rescue of ush null alleles. The restriction sites shown are EcoRI, BamHI, HindIII, NotI, which correspond to E, B, H, and N, respectively. (B) The sequence of the Ush protein corresponding to 1191 amino acids was predicted from both genomic and cDNA pU4.3 clones. The acidic region located in the amino-terminal part of Ush is boxed. Putative zinc fingers of both CCHC and CCHH type motifs are shown in grey, the CCHC ones are also boxed.

Figure 6

Structure of the Ush protein and comparisons with several other zinc finger proteins. (A) Location within the Ush protein of the acidic region, and the putative zinc finger motifs, CCHH and CCHC, are represented by different types of boxes. The CCHH and CCHC zinc finger motifs are numbered starting from the N part of the Ush protein, by numbers and letters, respectively. Homologous amino acids are shown in gray. (B) Comparison of the first CCHH zinc finger motif of Ush with the second one of the Zfy zinc finger proteins. The sequences represented correspond to amino acids 277–301 of Ush (USH1); amino acids 433–456 of the murine Zfy-1 (ZFY1_M; Ashworth et al. 1989); amino acids 450–474 of the human Zfy (ZFY_H; Palmer et al. 1990) and amino acids 448–472 of the murine Zfx1 (ZFX1_M; Mardon et al. 1990). (C) Comparison of the CCHC zinc finger motifs of Ush with several zinc finger proteins. cons refers to the consensus sequence presented by the CCHC zinc finger motifs of Ush. The sequences presented correspond to amino acids 210–232; 343–365; 728–750; 799–821; and 1121–1143 of Ush (USHa, USHb, USHc, USHd, and USHe, respectively); amino acids 1063–1085 of the Drosophila Schnurri (SHN_D; Grieder et al. 1995); amino acids 960–982 of the human PRDII-BF1 (PRDII_H; Fan and Maniatis. 1990); amino acids 651–675 of the human PRDI-BF1 (PRDI_H; Keller and Maniatis. 1991); amino acids 23–45 and 219–240 of the murine Evi-1 (Evi_M; Morishita et al. 1988) and amino acids 636–658 of the Drosophila Zfh-1 (ZFH1_D; Fortini et al. 1991). (–) A gap introduced to obtain maximal alignment.