BIOCHEMISTRY. For the article “Regulatory DNA required for vnd/NK-2 homeobox gene expression pattern in neuroblasts,” by Xiaoping Shao, Keita Koizumi, Neil Nosworthy, Dong-Ping Tan, Ward Odenwald, and Marshall Nirenberg, which appeared in number 1, January 8, 2002, of Proc. Natl. Acad. Sci. USA (99, 113–117; First Published December 18, 2001; 10.1073/ pnas.012584599), the authors note that the color quality in Figs. 1–3 was not adequate. Higher quality versions of the figures and their legends appear below.
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. A 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.