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First published online May 5, 2004
doi: 10.1242/10.1242/dev.01124

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A regulatory code for neurogenic gene expression in the Drosophila embryo

Michele Markstein^1,*,, Robert Zinzen^1,*, Peter Markstein², Ka-Ping Yee³, Albert Erives¹, Angela Stathopoulos¹ and Michael Levine^1,

¹ Department of Molecular and Cellular Biology, Division of Genetics and Development, 401 Barker Hall, University of California, Berkeley, CA 94720, USA
² Hewlett-Packard Laboratories, 1501 Page Mill Road, Palo Alto, CA 94304, USA
³ Computer Science Division Office, University of California, Berkeley, 387 Soda Hall #1776, Berkeley, CA 94720-1776, USA

View larger version (60K): [in a new window]   Fig. 1. Dorsal binding clusters identify regulatory DNAs. Diagrams on the left show the locations and sizes of five Dorsal binding clusters (depicted as blue boxes with sizes indicated below) identified in an earlier study (Markstein et al., 2002). In situ hybridization assays were performed to identify the expression profiles of the protein-coding genes (indicated as green boxes) located near the different clusters. Those genes found to be differentially expressed along the dorsal-ventral axis are shown in the middle column (`endogenous expression'). Genomic DNA fragments that encompass each of the five Dorsal-binding clusters were fused with a eve-lacZ reporter gene and expressed in transgenic embryos. Reporter gene expression (right column) was visualized by in situ hybridization using a digoxigenin-labeled lacZ antisense RNA probe. There is a close correspondence between the expression patterns of the endogenous genes and the staining patterns obtained with the fusion genes: sog (A) and CG12443 (B) are expressed throughout the neurogenic ectoderm; brk (C) is expressed in the ventral neurogenic ectoderm; and Phm (D) and Ady43A (E) are expressed in the mesoderm. Lateral views of cellularizing embryos oriented with anterior to the left and dorsal up are shown.

View larger version (65K): [in a new window]   Fig. 2. The coordinately expressed brk, vnd and rho enhancers share sequence motifs. Embryos in A, C and E express lacZ fusion genes containing the enhancer sequences indicated in B (brk, 498 bp), D (vnd, 348 bp) and F (rho NEE, 299 bp), respectively. Reporter gene expression was visualized by in situ hybridization, as described in Fig. 1. The three enhancers direct similar lateral stripes of lacZ expression. Each enhancer contains at least one copy of each of: CTGWCCY (indicated in green) and binding sites for Dorsal (black), Su(H) (red) and Twist (CA-core E-box, blue). Ventrolateral views of cellularizing embryos oriented with anterior to the left and dorsal up are shown.

View larger version (117K): [in a new window]   Fig. 3. The shared sequence motifs correspond to essential cis-regulatory elements. The shared sequence motifs in the vnd (A-C, 743 bp) and brk (E-H, 498 bp) enhancers were mutated as indicated, and the effects on enhancer activity were assayed by in situ hybridization as described in Fig. 1. Ventrolateral views of embryos oriented with anterior to the left and dorsal up are shown. All of the embryos (except D) are undergoing cellularization. (AC). A larger, more robust vnd enhancer than shown in Fig. 2 was used. The wild-type vnd enhancer directs lateral stripes of lacZ reporter gene expression (A). By contrast, point mutations that eliminate each of the two CACATGT motifs disrupt the activities of an otherwise normal vnd-lacZ fusion gene (B). Staining is restricted to the ventral-most regions of the neurogenic ectoderm, similar to the normal sim expression pattern (see Fig. 4). Mutations in the three CTGWCCY motifs in the vnd enhancer cause subtle changes in the lacZ staining pattern, including a slight narrowing and some irregularity in expression (C). (E-H). The embryos express different brk-lacZ fusion genes. The wild-type brk enhancer directs a staining pattern that is similar to the one produced by the vnd enhancer (E, compare with A). Mutations in the two CACATGT motifs disrupt the activities of the brk enhancer and cause a loss of lacZ staining, especially in the posterior half of the embryo (F, compare with E). Point mutations in the CTGWCCY motifs nearly abolish expression from an otherwise normal brk-lacZ fusion gene (G). Finally, mutations in the two Su(H)-binding sites cause a loss of expression in the posterior half of the embryo (H), similar to the altered pattern obtained with mutations in the Twist (CACATGT) binding sites (F). The transgenic embryo in D expresses a stripe2-NotchIC fusion gene that causes constitutive activation of Notch signaling in the stripe 2 region. The embryo was hybridized with a digoxigenin-labeled rho antisense RNA probe. Expression is slightly expanded in the region where the stripe2-NotchIC transgene is active (arrow).

View larger version (84K): [in a new window]   Fig. 4. Expression directed by newly identified fly and mosquito enhancers. The newly identified enhancers for vn (497 bp), sim (631 bp) and A. gambiae sim (976 bp) were fused to lacZ reporter genes. Embryos transgenic for these reporter constructs were analyzed by in situ hybridization, as described in Fig. 1. All embryos are depicted with anterior to the left. (A,C,E) Ventrolateral views of cellularizing embryos; (B,D,F) ventral views of gastrulating embryos. The vn enhancer drives expression in the ventral neurogenic ectoderm (A,B), similar to brk, vnd and rho (compare with Fig. 2A,C,E). The enhancer is located in the first intron of vn. The sim enhancer (C,D) drives expression in the mesectoderm, the ventral-most line of cells of the neurogenic ectoderm. The enhancer is located 5' of the sim gene. Weak and variable staining is also detected in more ventral regions of early embryos (C), possibly due to the loss of crucial Snail repressor sites. The Anopheles sim enhancer (E,F) drives irregular expression in the mesectoderm, similar to the pattern obtained with the Drosophila sim enhancer. The enhancer is located 5' of a putative sim ortholog. The relative arrangement and orientations of sequence motifs in the vn, sim and Anopheles sim enhancers are depicted in G: Dorsal motifs (black boxes), Su(H) motifs (red arrows), CA-Eboxes (CACATGT, dark-blue arrows) and CTGWCCY sites (green arrows). Additionally, the location of a sub-optimal Dorsal site (light gray box), a close relative to the CA-Ebox (CACATGG, light blue arrow), and two close matches to the CTGWCCY motif (CTGNCCY, light green arrows), are shown for the A. gambiae sim enhancer.

View larger version (20K): [in a new window]   Fig. 5. Model for differential gene expression in the neurogenic ectoderm. (A) Cross-section through a cellularizing embryo. The nuclear Dorsal gradient is shown with peak levels in ventral regions and lower levels in more lateral regions. The presumptive neurogenic ectoderm (NE) exhibits at least three distinct patterns of gene expression: sim and m8 are expressed only in the ventral-most line of cells in the NE, the mesectoderm; brk, vnd, rho and vn are expressed in the 5-6 cell wide ventral domain of the NE; and sog and CG12443 are expressed in broad lateral stripes throughout the NE. DE, dorsal ectoderm. (B) A stylized representation of the enhancers active in the NE. Enhancers active in the mesectoderm (e.g. sim) contain a large number of Su(H)-binding sites (red boxes), but few optimal dorsal sites (black boxes). By contrast, enhancers that direct broad expression throughout the NE (sog and CG12443) contain several optimal Dorsal sites, but no Su(H) sites. Enhancers that direct expression in an intermediate pattern, i.e. in ventral regions of the NE (rho, vnd, brk and vn), contain a mixture of high-affinity and low-affinity Dorsal sites, as well as a few Su(H) sites. Additionally, CA-Eboxes (CACATGT, blue boxes) and the CTGWCCY motif (not shown) are only found in the mesectodermal and ventral neurogenic ectodermal enhancers, and not in the enhancers driving broad expression in the NE. This implies that genes exhibiting overlapping expression patterns (e.g. sog and brk) are not activated solely by a gradient of nuclear Dorsal, but also by a variety of transcription factors, and also that they are activated in the same regions by different means.