Identification of the Control Region of Pancreatic Expression of Bmp4 In Vitro and In Vivo

<div><p>Bone morphogenetic protein 4 (Bmp4) was recently shown to be related to glucose homeostasis in mouse adult pancreas through the regulation of insulin production. We previously revealed the predominant expression of <i>Bmp4</i> in adult pancreas by <i>in vivo</i> imaging of transgenic mice. However, the control regions for predominant <i>Bmp4</i> expression in the adult pancreas are unclear. In this study, we established transgenic (Tg) mice that allow real time <i>in vivo</i> bioluminescence imaging of the enhancer/promoter activity of the <i>Bmp4</i> gene. Tg mice expressing firefly luciferase with a 7 kb upstream region and 5′-non-coding sequence (three exons and two introns) of the <i>Bmp4</i> gene showed pancreatic expression of bioluminescence, while the Tg mice bearing luciferase with the 7 kb upstream region alone did not show pancreatic expression of the reporter gene. Interestingly, pancreatic expression of bioluminescence was also present in Tg mice harboring the truncated promoter without exon IA and IB, indicating the presence of a cryptic promoter in front of exon II. Furthermore, the bioluminescence signal was not detected in embryonic pancreas, but increasing signals were observed in neonatal and infantile Tg mice depending on the genotypes observed. These results suggested that a novel mechanism of transcription is involved in pancreatic expression of the <i>Bmp4</i> gene.</p></div

Developmental expression of bioluminescence in Bmp4Luc Tg mice.

<p>(A) Bioluminescence signals in −6815/+4513 Bmp4Luc (line #17), (B) −6815/+618 Bmp4Luc (line #19) and (C) +1532/+4513 Bmp4Luc (Line #8) were determined at embryonic stage (E17.5), lactation stages (P5 and P16) and Weaning stage (P28 and beyond) using Xenogen IVIS Imaging System and the same reporter assay <i>in vivo</i> and <i>ex vivo</i> as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061821#pone-0061821-g002" target="_blank">Figure 2</a>. Embryonic bioluminescent images were obtained from laparotomy-treated embryonic bodies with the abdomen immersed in sterile PBS containing 0.3 mg/ml D-luciferin.</p

Luciferase activity of Bmp4Luc reporter constructs <i>in vitro</i>.

<p>(A) Mouse <i>Bmp4</i> is located on the long arm of mouse chromosome 14, and consists of four exons (IA, IB, II, III and IV) shown by boxes with two transcription start sites (TSS1, TSS2). Dark boxes show the coding sequence. The position of each element is counted from TSS1 as +1. Triangles denote splicing spans. (B) Luciferase activity of Bmp4Luc reporter constructs. NIT1 and NIH3T3 cells were transiently cotransfected with reporter plasmids and <i>Renilla</i> luciferase vector (pRL-TK) as a transfection control. The <i>x</i> axes show relative luciferase activities: firefly luciferase (FL) from the reporter plasmid was normalized to <i>Renilla</i> (RL) luciferase activity from the control vector. All data are the mean ± SE from three independent experiments. **Represents a significant difference (p<0.01) by Student’s <i>t</i>-test.</p

<i>In vivo</i> bioluminescent signals in Bmp4Luc Tg mice.

<p>(A–D) <i>In vivo</i> imaging assays of three representative lines of Bmp4Luc Tg mice [−6815/+4513 Bmp4Luc (line #17), −6815/+618 Bmp4Luc (line #19) and +1532/+4513 Bmp4Luc (Line #8)], and age matched (12–15-weeks-old). Transgenic mice were anesthetized with 2% isoflurane gas and subcutaneously injected with D-luciferin. Bioluminescent images were obtained by 1 min exposure in the imaging system. The minimum and maximum photons/second values for each figure are indicated in each rainbow bar scale. <i>Ex vivo</i> imaging assays were performed using dissected pancreas from transgenic mice immersed in sterile PBS containing 0.3 mg/ml D-luciferin, and subjected to imaging analysis. (E) The bioluminescence signals in each pancreas lines were quantified using Living Image software, and the intensity of luminescence is expressed as photons/second/cm²/steradian (p/s/cm²/sr) in individual transgenic mice and is indicated by individual color bars. The data are the mean ± SE from three independent experiments. **Represents a significant difference (p<0.01) by Student’s <i>t</i>-test.</p

Luciferase activity of truncated Bmp4Luc reporter constructs.

<p>(A) The truncated Bmp4Luc reporter was generated by restriction enzyme digestion as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061821#s4" target="_blank">Materials and Methods</a> to determine luciferase activity as described in Fig. 1B. (B) Bioluminescence signals were detected in +2.9 kb-Bmp4Luc Tg mice (#30) using the same reporter assay <i>in vivo</i> and <i>ex vivo</i> as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061821#pone-0061821-g002" target="_blank">Figure 2</a>. **Represents a significant difference (p<0.01) by Student’s <i>t</i>-test.</p

Establishment Bmp4Luc Tg mice.

*<p>Representative lines used for <i>in vivo</i> imaging. (n), number.</p

Establishment of 7 kb-Bmp4Luc transgenic mice.

<p>(A) Genomic PCR of transgenic mouse lines (#1, #8 and #17) produced by pro-nuclear microinjection of linearized p7kb-Bmp4Luc. Arrowheads indicate primer sets in the left panel. Template DNA was prepared from tails of each transgenic line and from wild-type mice (Non-Tg) and plasmids (pBmp4-Luc). (B) Fluorescence in situ hybridization (FISH) assays were performed using fibroblast cells isolated from the tail of each transgenic mouse line. p7kb-Bmp4Luc plasmid DNA was used as a probe (yellow signals with arrows). Chromosomal DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue).</p

Number of Tg mice expressing bioluminescence signals in pancreas and skin during pancreatic maturation against the number of tested mice.

<p>Luciferase activity in the pancreas was detected by <i>ex vivo</i> imaging of upper 10⁵ (p/s/cm²/sr) and in skin by <i>in vivo</i> imaging of upper 10⁵ (p/s/cm²/sr) using the Xenogen IVIS Imaging System. The numbers of bioluminescence positive lines observed from the total tested are shown. Colored boxes represent a positive bioluminescence signal.</p

In vivo bioluminescent signals in p7 kb-Bmp4Luc transgenic mice.

<p>(A) In vivo imaging assays of the three lines of transgenic mice (#1, #8 and #17). Transgenic mice were anesthetized with 2% isoflurane gas and intraperitoneally injected with D-luciferin. Bioluminescent images were obtained by 1 min exposure in the imaging system. The minimum and maximum photons/second values for each figure are indicated in each rainbow-colored bar scale. Strong bioluminescent signals were detected in the upper abdomen (black arrows) in all three lines in vivo. (B) The strong signal in the upper abdomen was confirmed to be localized to the pancreas (black arrow), by abdominal exploration in a transgenic mouse injected with D-luciferin. (C) Ex vivo imaging assays. Dissected tissues, spleen (S), liver (Li), pancreas (P), lung (Lu) and kidney (K) from wild-type (upper part of the dish) and transgenic mice (bottom part of the dish) were immersed in sterile PBS containing 0.3 mg/ml D-luciferin, and subjected to imaging analysis. (D) Western blot analysis of various tissues from transgenic mice, using antibodies to murine Bmp4, luciferase and α-tubulin.</p

Bmp4 and luciferase proteins in pancreatic islets.

<p>Immunohistochemical analysis of pancreas (A–D) and liver (E–H) of wild-type (A, C, E, G) and transgenic mice (B, D, F, H) was performed using antibodies to Bmp4 (A, B, E, F) and luciferase (C, D, G, H). Bmp4-positive cells and luciferase-positive cells are shown as brown cells (arrows) in pancreatic islets. Scale bar shows 25 μm.</p