Ind represses msh expression in the intermediate column of the Drosophila neuroectoderm, through direct interaction with upstream regulatory DNA

Abstract

The Drosophila neurectoderm is initially subdivided across the dorsoventral (DV) axis into three domains that are defined by the expression of three homeodomain containing proteins. These are from ventral to dorsal: Ventral nervous system defective (vnd), Intermediate neuroblasts defective (ind) and Muscle segment homeobox (msh). This is remarkably similar to the distribution of the orthologous homeodomain proteins in the developing neural tube of mice and Zebrafish. This pattern is partially governed by a 'ventral dominance' mechanism, in which Vnd represses ind and Ind represses msh. A major unanswered question in this process is: How does Ind direct positioning of the ventral border of msh expression. Toward this goal, we have identified regulatory DNA essential for expression of msh in the early neurectoderm. In addition, we demonstrated that Ind acts directly in this element by a combination of genetic and molecular experiments. Specifically, expression is expanded ventrally in ind mutant embryos and Ind protein directly and specifically bound to the msh regulatory DNA, and this interaction was required to limit the ventral boundary of msh expression.

Figure 1

Identification of Msh regulatory DNA. Top Line A) Diagram of the genomic region upstream of, and including msh coding sequences. Green boxes indicate regions of conserved sequence among Drosophila species. Approximate distances from the coding sequences are indicated for the end of each conserved block. Red boxes indicate Twist binding regions identified by ChIP experiments (Sandmann et al., 2007) (Zeitlinger et al., 2007). Blue boxes are a schematic of msh exons. Black scale bar represents approximately 1kb. B-E) lacZ mRNA in msh2-lacZ embryos at stages of increasing age. B) Stage 6 C) Stage 9 D) Stage 11 E) stage 14. All embryos are ventro-lateral views, anterior is up.

Figure 2

Sequences in the first intron of msh control expression in mesodermal precursors. A) stage 11 mshI-lacZ embryo, lacZ mRNA. B) WT embryo stage 11, msh mRNA. Arrows indicate the positions of lateral mesodermal precursor cells. C-E) Doubly labeled, stage 11 mshI-lacZ embryo, β—gal protein brown and Msh protein in blue. D-E) High magnification images of double label embryos arrows show that domains are partially overlapping. F) late stage mshI-lacZ embryo, lacZ mRNA. Expression is detected in glial cells. A-C) Embryos are lateral views, anterior is up. D-E) Embryos are ventral views, anterior is up.

Figure 3

Msh2lacZ contains sequences necessary for repression by Ind in the intermediate column. A-D) Wild type embryos. A) Msh2-lacZ mRNA Stage 6 B) Msh2- lacZ mRNA Stage 9 C) Msh2-lacZ mRNA (blue), Ind protein (brown) stage 9. D) Msh2-lacZ mRNA (blue), Ind protein (brown) stage 11. E) ind^16.2 embryo Msh2-lacZ mRNA stage 6. F) ind^16.2 embryo Msh2-lacZ mRNA stage 9. G-H) High magnification images of stage 9/10 WT (G) and ind^16.2 (H) embryos arrows emphasize change in distance between msh2lacZ mRNA stripes. I) Msh2-lacZ mRNA in KrGal4x UAS ind embryo stage 10. J) Msh2-lacZ mRNA in vnd; ind Msh2-lacZ embryo stage 10. K) Msh2-lacZ mRNA in KrGal4x UAS Vnd embryo stage stage 9/10. All embryos are ventral views except A and E which are lateral views and I and K which are ventro-lateral views. Anterior is up in all cases.

Figure 4

A 699bp fragment contains the minimal element necessary for neurectoderm expression. A) Schematic illustrating the subdivision of the msh2 element to further define the minimal element. Top shows sizes of fragments a, b, c, and d. bottom shows approximate position of msh2 element relative to the coding sequences. B) Msh2a-lacZ mRNA stage 6. C) Msh2a-lacZ mRNA stage 9. D) Msh2a-acZ mRNA stage 10. E) Msh2a-lacZ mRNA stage 11. F) Msh2b-lacZ mRNA stage 6. All embryos are positioned anterior up.

Figure 5

The Ind homeodomain binds to the msh2a element in a sequence specific manner. A) sequence of msh2a region use as probe for DNA binding in panels B and C. Possible Ind sites are highlighted in grey. B) IndHD binds to a fragment containing the first two putative Ind binding sites. C) Ind binds to and shifts DNA containing mutated site 1 (lanes 4-6) but not mutated site 2 (lanes 1-3), or both (lanes 7-9). D) sequence of msh2a region use as probe for DNA binding in panels E and F. Possible Ind sites are highlighted in grey. E) IndHD binds to a fragment containing putative Ind binding sites three and four. F) Ind binds to and shifts DNA containing mutated site 3 (lanes 1-3) but not mutated site 4 (lanes 4-6), or both (lanes 7-9).

Figure 6

Intact Ind bindings sites in the msh regulatory DNA are essential for repression of msh2a-lacZ in the intermediate column. A-B) expression of the WT msh2a-lacZ message in stage 9 embryos. A) lateral view B) ventral view. C) WT msh2a embryo labled for Ind protein and βGal protein. D-E) Expression of the msh2amut lacZ message in stage 9 embryos. D) lateral view E) ventral view. Arrows are added to emphasize the change in distance between the stripes of lacZ expression. F) msh2a mut-lacZ embryo labeled for Ind protein and βGal protein. Arrows point to cells that appear doubly labeled for both Ind and βGal, illustrating that there are regions of over-lap between the Ind and βGal patterns.

Figure 7

Msh activity is not required for initiation of maintenance of msh expression. A-C) Wild type embryos, msh mRNA. A) stage 6. B) stage 9. C) stage 10. D-F) msh^SOD54 embryos, msh mRNA. D) stage 6. E) stage 9. F) stage 10. G-I) msh^Δ68 embryos msh2-lacZ mRNA. G) stage 6. H) stage 9. I) stage 10. All embryos are ventrolateral views, anterior is up.