Regulatory DNA required for vnd/NK-2 homeobox gene expression pattern in neuroblasts

Abstract

Vnd/NK-2 protein was detected in 11 neuroblasts per hemisegment in Drosophila embryos, 9 medial and 2 intermediate neuroblasts. Fragments of DNA from the 5'-flanking region of the vnd/NK-2 gene were inserted upstream of an enhancerless betagalactosidase gene in a P-element and used to generate transgenic fly lines. Antibodies directed against Vnd/NK-2 and beta-galactosidase proteins then were used in double-label experiments to correlate the expression of beta-galactosidase and Vnd/NK-2 proteins in identified neuroblasts. DNA region A, which corresponds to the -4.0 to -2.8-kb fragment of DNA from the 5'-flanking region of the vnd/NK-2 gene was shown to contain one or more strong enhancers required for expression of the vnd/NK-2 gene in ten neuroblasts. DNA region B (-5.3 to -4.0 kb) contains moderately strong enhancers for vnd/NK-2 gene expression in four neuroblasts. Hypothesized DNA region C, whose location was not identified, contains one or more enhancers that activate vnd/NK-2 gene expression only in one neuroblast. These results show that nucleotide sequences in at least three regions of DNA regulate the expression of the vnd/NK-2 gene, that the vnd/NK-2 gene can be activated in different ways in different neuroblasts, and that the pattern of vnd/NK-2 gene expression in neuroblasts of the ventral nerve cord is the sum of partial patterns.

Figure 1

Identification of Vnd/NK-2 expressing NBs in the ventral nerve cord. To identify the NBs expressing Vnd/NK-2, the enhancer trap NB marker Drosophila lines en-lacZ, wg-lacZ, and hkb⁵⁹⁵³ were used. In each line, β-Gal is expressed in identified NBs. Antibodies to Vnd/NK-2 protein (red) and β-Gal protein (green) were used for double staining. (A) Double-stained en-lacZ embryo at stage 11; β-Gal is expressed in the engrailed pattern in posterior compartment cells. NBs 1-2, 6-1, 7-1, and 7-2 were shown to express both Vnd/NK-2 and β-Gal proteins. The bar corresponds to 5 μm. (B) Double-stained wg-lacZ embryo at stage 11; NBs 5-1 and 5-2 are Vnd/NK-2 and β-Gal positive. (C) Double-stained hkb⁵⁹⁵³ embryo at stage 11; only NB 2-1 contains both Vnd/NK-2 and β-Gal proteins. (D) Schematic summary of the identity of Vnd/NK-2 positive NBs. At stage 11, the following nine NBs (orange centers) were identified that are Vnd/NK-2 positive per hemisegment: 1-2, 2-1, 3-1, 4-1, 5-1, 5-2, 6-1, 7-1, and 7-2. MP-2 and 1-1 also express Vnd/NK-2 earlier in development and are not shown here. Light blue, en-lacZ positive NBs; green, wg-lacZ positive NBs; purple, hkb⁵⁹⁵³ positive NBs (29).

Figure 2

Endogenous Vnd/NK-2 expression. β-Gal expression regulated by the −5.3/+0.35-kb DNA fragment from the 5′-upstream region of the vnd/NK-2 gene in neuroectoderm compared with NBs. Red, Vnd/NK-2 protein; green, β-Gal protein. (A) Neuroectoderm layer of a double-labeled embryo at early stage 11. A column of neuroectodermal cells, one cell wide, that contain NK-2 protein is seen on each side of the mesectoderm. The arrowhead points to an NB that is close to the neuroectoderm layer (possibly 6-1) that has a higher level of β-Gal expression than that of neuroectodermal cells. (B) NB layer of double-labeled embryo; β-Gal expression pattern (green), directed by vnd/NK-2 (−5.3/+0.35)-β-gal construct almost completely overlaps the endogenous Vnd/NK-2 pattern; however, Vnd/NK-2 but not β-Gal was expressed in NB 7-2 (arrow). The bar corresponds to 5 μm.

Figure 3

Regulatory DNA required for Vnd/NK-2 pattern formation in NBs. Fragments of DNA from the 5′-upstream region of the vnd/NK-2 gene, subcloned in the 5′-flanking region of an enhancerless β-gal gene in a P-element, were used to generate transgenic lines of Drosophila. Antibodies directed against Vnd/NK-2 protein (red) or β-Gal (green) were used to double-label NBs in the ventral nerve cord of stage 11 transgenic embryos. The neuroblasts are identified in the panels on the right. An NB with an orange center surrounded by a green ring expressed both Vnd/NK-2 and β-Gal. An NB that expressed Vnd/NK-2, but not β-Gal, is shown in red. Cells that ectopically express β-Gal are shown with white centers surrounded by green. (A) β-Gal expression regulated by the −5.3/−2.8-kb DNA fragment from the 5′-upstream region of the vnd/NK-2 gene in a transgenic embryo. The bar corresponds to 5 μm. (B) β-Gal expression regulated by the −4.7/−2.8-kb DNA fragment from the 5′-upstream region of the vnd/NK-2 gene. The arrow and arrowheads indicate cells (E1 and E2) that ectopically express β-Gal. (C) β-Gal expression regulated by the −4.0/−2.8-kb DNA fragment from the 5′-upstream region of the vnd/NK-2 gene. Arrowheads indicate the ectopic expression of β-Gal in E1 and E3 cells. (D) β-Gal expression regulated by the −5.3/−3.5-kb DNA fragment from the 5′-upstream region of the vnd/NK-2 gene. β-Gal is expressed strongly in two NB, 1-2 and 3-1, and weakly in NB 2-1 and NB 4-1. (E) β-Gal expression directed by the −5.3/−4.0-kb DNA fragment from the 5′-upstream region of the vnd/NK-2 gene in a transgenic embryo. The four NBs that express both β-Gal and Vnd/NK-2 shown in D also express both proteins in E; however, β-Gal expression in the embryo shown in E is weaker than the expression shown in D. The levels of expression of β-Gal in NBs 1-2 and 3-1 are greater than the expression found in NBs 2-1 and 4-1.

Figure 4

Summary of the analysis of vnd/NK-2 regulatory DNA. Five DNA fragments from the 5′-upstream region of the vnd/NK-2 gene were subcloned in the 5′-flanking region of an enhancerless β-gal gene in a P-element. The constructs were used to generate transgenic lines of Drosophila. The number of transgenic lines of flies generated for each construct is shown (enclosed by parentheses). DNA region A, −4.0 to −2.8 kb, is sufficient to direct Vnd/NK-2 expression in the following ten NBs: 1-1 (transient expression, not shown), 1-2, 2-1, 3-1, 4-1, 5-1, 5-2, 6-1, 7-1, and MP-2. In addition, region A activates ectopic Vnd/NK-2 expression in two cells, E1 and E3. DNA region B (B1 and B2, −5.3 to −4.0 kb) contains nucleotide sequences that activate the vnd/NK-2 gene in four NBs as follows: 1-2, 2-1, 3-1, and 4-1. DNA region B2 (−4.7 to −4.0 kb) activates the vnd/NK-2 gene ectopically in cell E2 and represses the vnd/NK-2 gene in E3. DNA region B1 (−5.3 to −4.7 kb) contains nucleotide sequences that repress the ectopic expression of the vnd/NK-2 gene in E1 and E2. Hypothesized DNA region C, whose location has not been identified, activates the vnd/NK-2 gene in NB 7-2. ML rep (−3.6 to −3.1 kb) corresponds to the region of DNA in the 5′-flanking region of the vnd/NK-2 gene that Estes et al. (18) have shown is required for midline repression of the vnd/NK-2 gene mediated indirectly by the Sim protein in mesoectodermal cells.