Functional dissection of AS071 using deletion constructs.

<p>(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 <i>lacZ</i> reporter gene (grey box) followed by the SV40 polyA signal (orange box). Resulting constructs were linearized with <i>Xho</i> I and <i>Not</i> 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 <i>lacZ</i> expression in each expression domain is indicated. Efficiency denotes the number of embryos showing <i>lacZ</i> expression per those carrying the transgene. (C) (Left) <i>LacZ</i> expressions directed in the diencephalic region by the deletion constructs. Note that all constructs lacking the SINE sub-element (red box) could not direct <i>lacZ</i> 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.</p

<i>LacZ</i> expression in AS071-transgenic mice and comparison of <i>Fgf8</i> expression between mouse and chicken.

<p>(A) <i>LacZ</i> expression pattern in developing mouse diencephalon directed by AS071 in the transgenic Line C. X-Gal staining for β-galactosidase activity in AS071-<i>lacZ</i> transgenic mouse embryos through E9.5–E15.5 shows a dynamic pattern of <i>lacZ</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043785#pone.0043785.s002" target="_blank">Figure S2</a>. 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 <i>Fgf8</i> 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.</p

Conservation of the sequence and surrounding gene synteny of AS071.

<p>(A) The human AS071 (chr10∶103,356,749–103,357,337, GRCh37/hg19) in the UCSC Genome Browser (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>). 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043785#pone.0043785.s001" target="_blank">Figure S1</a>. (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.</p

A model of domain-specific expression of diencephalic <i>Fgf8</i> by AS071.

<p>Spatiotemporally restricted <i>Fgf8</i> 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.</p

AS3_9 locus and its enhancer activity in the frontonasal region.

<p>(A) Location of human AS3_9 and surrounding genes. (B) The phastCons conservation track of AS3_9 in mouse (UCSC Genome Browser; mm10); the blue, yellow, and green bars denote the AmnSINE1, X6b_DNA, and MER117 regions, respectively. The construct used to produce AS3_9-<i>lacZ</i> mice is illustrated below. (C) Whole-mount (left), frontal view of the head (top right), and ventral view of the upper jaw (bottom right) showing <i>lacZ</i> expression in AS3_9-<i>lacZ</i> E13.5 embryos. (D) Coronal section of an AS3_9-<i>lacZ</i> E14.5 embryo. (E) ISH for <i>wnt5a</i> in E14.5 wild type. Arrowheads indicate the palatal shelves (C–E). MN, medial nasal process; LN, lateral nasal process; Max, maxillary process; Man, mandibular process. Scale bar (C–E): 0.5 mm.</p

<i>Wnt5a</i> expression in E14.5 coronal sections from the anterior and intermediate regions of the oral cavity in one wild-type and three AS3_9-ko embryos (#1–3).

<p>(A) Anterior region of a wild-type embryo. (B-D) Anterior region of three AS3_9-ko embryos. (E) Intermediate region of a wild-type embryo. (F-H) Intermediate region of three AS3_9-ko embryos. Arrowheads denote palatal shelves. Scale bar: 0.5 mm.</p

Functional dissection of AS3_9 enhancer.

<p>Top left: Conservation track from the UCSC Genome Browser (mm10) and TE regions in mouse. Shown are nine constructs (a–j) and their enhancer activity in the maxillary and mandibular processes, nasal processes, and palatal shelves (right). Lateral view (a'–j') and ventral view (a''–j'') of the upper jaw of E13.5 transgenic embryos harboring each deletion construct (a–i) and the construct with a mutated Msx1-binding site (j). <i>LacZ</i> expression was clearly observed in the maxillary and mandibular processes (yellow arrowheads), nasal processes (green arrowheads), and palatal shelves (red arrowheads). Scale bar: 1 mm.</p

<i>Wnt5a</i> expression in E14.5 coronal sections from the anterior and intermediate regions of the oral cavity in one wild-type and three AS3_9-ko embryos (#1–3).

<p>(A) Anterior region of a wild-type embryo. (B-D) Anterior region of three AS3_9-ko embryos. (E) Intermediate region of a wild-type embryo. (F-H) Intermediate region of three AS3_9-ko embryos. Arrowheads denote palatal shelves. Scale bar: 0.5 mm.</p

Functional dissection of AS3_9 enhancer.

<p>Top left: Conservation track from the UCSC Genome Browser (mm10) and TE regions in mouse. Shown are nine constructs (a–j) and their enhancer activity in the maxillary and mandibular processes, nasal processes, and palatal shelves (right). Lateral view (a'–j') and ventral view (a''–j'') of the upper jaw of E13.5 transgenic embryos harboring each deletion construct (a–i) and the construct with a mutated Msx1-binding site (j). <i>LacZ</i> expression was clearly observed in the maxillary and mandibular processes (yellow arrowheads), nasal processes (green arrowheads), and palatal shelves (red arrowheads). Scale bar: 1 mm.</p

Expression of the CaMKIIβ throughout the brain by AAV-PHP.eB.

Volume-rendered and single-plane images of the brain expressing H2B-mCherry under hSyn1 promoter by the AAV (mCherry, green) counterstained with RD2 (red). A volume-rendered image is shown in the center. Single-plane and magnified images are shown for cerebral cortex, thalamus, hippocampus, midbrain, cerebellum, striatum, and olfactory bulb. Scale bar in the center image, 3 mm; other scale bars, 100 μm. AAV, adeno-associated virus; CaMKIIβ, calmodulin-dependent protein kinase IIβ; hSyn1, human synapsin-1. (TIFF)</p