Role of homeodomain protein binding region in the expression of Drosophila proliferating cell nuclear antigen gene: analysis with transgenic flies

Abstract

The regulatory region of Drosophila proliferating cell nuclear antigen (PCNA) gene consists of a promoter region (-168 to +24 with respect to the transcription initiation site) and an upstream region containing three homeodomain protein binding sites (HDB) (-357 to -165). The PCNA gene regulatory regions with HDB (-607 to +137) or without HDB (-168 to +137) were fused with the lacZ and transgenic flies were established by P-element-mediated transformation. Male transgenic flies were crossed with wild-type females, and zygotic expression of the lacZ was monitored by quantitative beta-galactosidase assay, at various stages of development. Expression of the lacZ was high in embryos, first and second instar larvae, and adult females, and low at other stages of development. Only a marginal difference in expression was observed between flies carrying the homeodomain protein binding region and those not carrying it. Spatial pattern of the lacZ expression in the embryo visualized by immunostaining with the anti-lacZ antibody was similar to the distribution of the endogenous PCNA protein. Here, too, only a marginal difference was observed between transgenic flies carrying two different constructs of the PCNA lacZ. In genetic crossing experiments of transgenic flies with those carrying mutation in homeobox genes, no significant change in the lacZ expression pattern was observed. However, when male transgenic flies were crossed with female flies homozygous for a torso gain-of-function allele, repression of the lacZ expression was observed in the central region of the embryo. Because these local changes in the lacZ expression depend on the homeodomain protein binding region, unidentified homeodomain proteins are probably involved. Our results suggest that the promoter region is practically sufficient for expression of the PCNA gene and that the homeodomain protein binding region functions as a silencer when torso is activated ectopically.

FIG. 1

Distribution of PCNA protein in wild-type embryos. Whole-mount specimens of fixed embryos were immunostained with an anti-PCNA antibody. Embryos are oriented so that anteriors are to the left and ventrals are to the bottom [except in (D)]. Staging is according to Campos-Ortega and Hartenstein (1985). (A) Surface view of the syncytial blastoderm embryo (stage 4, 1320–1410 h). (B) Internal focal view of the blastoderm embryo (stage 5, 1410–1450 h), pole cells (pc). (C) Internal focal view of the early gastrulating embryo (stage 6, 1450–1500 h), ventral furrow (vf). (D) Ventral surface view of the early gastrulating embryo (stage 6). (E) Surface view of the embryo of the stage beginning of germ band extension (stage 7, 1500–1510 h), cephalic furrow (cf). (F) Internal focal view of the embryo shown in (E), anterior midgut primordium (am), proctodeum primordium (pr), posterior midgut primordium (pm). (G) Internal focal view of the stage 8 embryo (1510–1540 h) (germ band elongation), mesoderm (ms), ectoderm (ec). (H) Internal focal view of the stage 9 embryo (1540–1620 h). (I) Surface view of the stage 10 embryo (1620–1720 h). (J) Internal focal view of stage 10 embryo, stomodeum (sto). (K) Internal focal view of the early stage 11 embryo. (L) Stage 10 ebmbryo treated with normal rabbit IgG as a primary antibody.

FIG. 2

Maternal contribution to PCNA lacZ expression. Embryos from w males × p5′-607DPCNAlacZW8HS females (A–E) or w males × p5′-168DPCNAlacZW8HS females (F–J), as indicated at the top, were immunostained with anti-lacZ antibody. (A,F) Surface views of syncytial blastoderm embryos (stage 4, 1320–1410 h). (B,G) Surface views of cellular blastoderm embryos (stage 5, 1410–1450 h). (C,H) Surface views of stage 7 embryos (1300–1510 h), cephalic furrow (cf). (D,I) Internal focal views of stage 8 embryos (1510–1540 h). (E,J) Internal focal views of stage 10 embryos (1620–1720 h), stomodeum (sto).

FIG. 3

Zygotic expression of PCNA lacZ in embryos. Embryos from p5′-607DPCNAlacZW8HS males × w females (A–J) or p5′-168DPCNAlacZW8HS males × w females, as indicated at the top, were immunostained with an anti-lacZ antibody. Surface views of (A,K) syncytial blastoderm embryos (stage 4, 1320–1410 h), (B,L) cellular blastoderm embryos (stage 5, 1410–1450 h), (C,M) early gastrulating embryos (stage 6, 1450–1500 h), (D,N) embryos at the beginning of germ band elongation (early stage 7, 1500–1510 h), and (E,O) embryos at the early phase of germ band elongation (late stage 7) are shown; pole cells (pc) and cephalic furrow (cf). (F,P) Internal focal views of embryos shown in (E,O), anterior midgut primordium (am), proctodeum primordium (pr), posterior midgut primordium (pm), and aminoserosa (as). Internal focal views of (G,Q) stage 8 embryos (1510–1540 h), (H,R) stage 9 embryos (1540–1620 h), (I,S) stage 10 embryos (1620–1720 h), and (J,T) stage 11 embryos (1720–1920 h) are shown; mesoderm (ms), ectoderm (ec), stomodeum (sto).

FIG. 4

Expression of PCNA lacZ during development. Male transgenic flies were crossed with female wild-type flies and extracts were prepared from Drosophila bodies at various stages of development. The β-galactosidase activities in the extracts are expressed as absorbance units per h per mg protein. Black bars indicate the averaged value of eight independent transgenic strains carrying p5′-607DPCNAlacZW8HS. Hatched bars indicate the averaged value of six independent strains carrying the p5′-168DPCNAlacZW8HS. Deviation between independent strains is also indicated.

FIG. 5

Expression of PCNA lacZ in tor ^RL3 mutant embryos. Embryos from p5′-607DPCNAlacZW8HS males × tor ^RL3 females (A–D) or p5′-168DPCNAlacZW8HS males × tor ^RL3 females (E–H), as indicated at the top of each panel, were immunostained with anti-lacZ antibody. Surface views of 3–6-h embryos are shown. Stages of the embryos shown in (D) and (H) are more advanced than those shown in (A–C) and (E–G). In (B) and (F), indicated parts of (A) and (E) are shown in higher magnifications; ventral furrow (vf), posterior midgut primordium (pm), proctodeum primordium (pr), anterior midgut primordium (am).

FIG. 6

Distribution of PCNA protein in tor ^RL3 mutant embryos. Whole-mount specimens of fixed embryos were immunostained with an anti-PCNA antibody. Embryos are oriented so that anterior aspects are to the left and ventral areas are down [except (B) and (G)]. (A) Surface view of the early gastrulating wild-type (Canton S) embryo (stage 6, 1420–1500 h). (B) Ventral surface view of the early gastrulating wild-type embryo (stage 6). (C) Surface view of the wild-type embryo at initiation of germ band elongation (stage 7, 1500–1510 h). (D–I) Surface views of 2–6-h tor ^RL3 embryos. Ventral surface view is shown in (G). Stage of the embryo shown in (H) is more advanced than those in (D–G). In (F) and (I), indicated parts of (E) and (H) are shown in higher magnifications; cephalic fold (cf), pole cell (pc), ventral furrow (vf).