<i>phd3</i> is expressed in infected macrophages during early stage Mm pathogenesis in a Hif-1α dependent manner.

<p>(A) Fluorescent confocal micrographs of 2 examples of infected areas prior to granuloma formation of 1 dpi embryos (upper and lower panels). <i>phd3</i> expression was detected by GFP levels, in green, using the <i>Tg(phd3:GFP)i144</i> transgenic line. Mm mCherry is shown in the red channel. Increased levels of <i>phd3</i>:GFP expression were detectable in cells associated with infection. (B) Fluorescent confocal micrographs of 2 granulomas at 6 dpi (upper and lower panels). Only low levels of <i>phd3</i>:GFP expression were detectable in areas of infection. The low level of GFP is illustrated in the upper panel where the auto-fluorescence of a pigment cell (white arrowhead) is brighter than the phd3:GFP expression. (C) Fluorescent confocal micrographs of 2 embryos with Mm infected macrophages at 1 dpi. The <i>phd3</i>:GFP line was outcrossed to the <i>mpeg1</i>:mCherry line to show co-localization with infected macrophages. (D) <i>phd3</i>:GFP embryos were injected at the 1 cell stage with dominant negative <i>hif-1αb</i> RNA (DN1) or phenol red (PR) as a negative control. 60 embryos of each were screened for <i>phd3</i>:GFP expression using confocal microscopy and the 3 brightest areas of <i>phd3</i>:GFP expression were imaged and showed co-localization with Mm infection. In the DN1 group GFP laser levels and confocal settings were increased until background green fluorescence was visible showing no specific co-localisation with Mm.