Figure 1.
Conservation of the sequence and surrounding gene synteny of AS071.
(A) The human AS071 (chr10∶103,356,749–103,357,337, GRCh37/hg19) in the UCSC Genome Browser (http://genome.ucsc.edu/). The red bar corresponds to the AmnSINE1-derived region (homologous to the AmnSINE1 consensus sequence, 204 bp), and the black bar shows the region conserved among mammals (590 bp). Sequence alignment of the region is shown in Figure S1. (B) The conserved gene synteny of the 1-Mb region around the locus among human (chr10∶103,000,000–104,000,000, GRCh37/hg19), mouse (chr19∶45,310,000–46,310,000, NCBI37/mm9), dog (chr28∶16,900,000–17,900,000, Broad/canFam2), and opossum (chr1∶110,001,000–111,001,000, Broad/monDom5). Note the inversion in this region in opossum. Gene annotations are based on the human assembly in the UCSC Genome Browser.
Figure 2.
LacZ expression in AS071-transgenic mice and comparison of Fgf8 expression between mouse and chicken.
(A) LacZ expression pattern in developing mouse diencephalon directed by AS071 in the transgenic Line C. X-Gal staining for β-galactosidase activity in AS071-lacZ transgenic mouse embryos through E9.5–E15.5 shows a dynamic pattern of lacZ expression in the diencephalic domain. An ectopic expression in midbrain peculiar to this line was observed at E15.5. (a–g) Dorso-frontal views of whole-mount stained dissected brains. (a’–g’) Lateral views. The left telencephalon was removed. (a”–g”, e”’–g”’) Sagittal sections. The expression profiling of all three lines is shown in Figure S2. Scale bars: 0.5 mm (a–c, a’–c’), 1.0 mm (d–g, d’–g’), 0.4 mm (a”–g”, e”’–g”’). (B) Comparison of spatiotemporal Fgf8 expression between mouse and chicken by in situ hybridization. The mouse E10.5, E12.5 and E14.5 stages correspond to chicken HH21, HH23 and HH30, respectively. Colored arrowheads indicate as follows: green, dorsal midline of the diencephalon; blue, lateral wall of the diencephalon; red, ventral midline of the hypothalamus; black, anterior neural ridge; white, optic recess.
Figure 3.
Functional dissection of AS071 using deletion constructs.
(A) Genomic region surrounding the mouse AS071 in the UCSC Genome Browser (chr19∶45,641,837–45,642,422, NCBI37/mm9) and schematic representation of the transgene constructs. AS071 was split into four sub-elements for the deletion analyses: CNE1a, blue box; CNE1b, green box; SINE, red box; CNE2, turquoise box. The fragments with various combinations of AS071 sub-elements were amplified from the AS071-HSF51 construct and re-introduced in the HSF51 vector harboring the mouse heat-shock protein promoter (black box) and bacterial lacZ reporter gene (grey box) followed by the SV40 polyA signal (orange box). Resulting constructs were linearized with Xho I and Not I before microinjection. (B) Results of the enhancer analysis using the AS071-deletion constructs. The letters a–j on the left correspond to those in (C). Schematic diagrams of the organization of each construct are shown. The presence (+) or absence (–) of lacZ expression in each expression domain is indicated. Efficiency denotes the number of embryos showing lacZ expression per those carrying the transgene. (C) (Left) LacZ expressions directed in the diencephalic region by the deletion constructs. Note that all constructs lacking the SINE sub-element (red box) could not direct lacZ expression in the ventral midline of the hypothalamus. Arrowheads indicate as follows: green, dorsal midline of the diencephalon; blue, lateral wall of the diencephalon; red, the ventral midline of the hypothalamus. (Right) The constructs lacking CNE1b (green box) show no enhancer activity in the diencephalon. (D) Summary of the functional dissection of AS071. Whether each AS071-enhancer sub-element showed activity in a particular region is indicated by + or –. The color of each element corresponds to the associated diencephalic domain shown in the illustration. DD, dorsal midline of the diencephalon; LD, lateral wall of the diencephalon; VMH, ventral midline of the hypothalamus.
Figure 4.
Comparison of spatiotemporal lacZ expression pattern between the AS071-ΔSINE-and AS071-transgenic lines.
LacZ expression in AS071-ΔSINE-transgenic embryos shows exactly same pattern as that in AS071-transgenic embryos between E9.5–E14.5 except in the ventral midline of the hypothalamus (red broken-line triangle), indicating the role of the SINE sub-element in determining domain-specificity of the activity to the ventral midline of the hypothalamus. (a–f) Dorso-frontal views of whole-mount stained dissected brains. (a’–f’) Lateral views. The left telencephalon was removed. (a”–f”, e”’–f”’) Sagittal sections. Colored arrowheads represent as follows: green, dorsal midline of the diencephalon; blue, lateral wall of the diencephalon; red, the ventral midline of the hypothalamus. Scale bars: 0.5 mm (a–c, a’–c’), 1.0 mm (d–g, d’–g’), 0.4 mm (a”–g”, e”’–g”’).
Figure 5.
A model of domain-specific expression of diencephalic Fgf8 by AS071.
Spatiotemporally restricted Fgf8 expression in the developing diencephalon by AS071 is accomplished through the interaction of activator proteins that cooperatively bind to each sub-element. The coactivator protein (purple box) mediates the interaction between the activator protein (yellow box), which is ubiquitously expressed in the diencephalon, and other domain-specific activator proteins, as indicated by the colored shapes. Each domain-specific activator protein needs to associate with the core element in CNE1b to effect enhancer activity in each diencephalic domain.