<i>Gng5^−/−</i> mutants show loss of second heart field and reduced expression of proliferative markers.

<p><b>Panels A–C</b>, control and mutant embryos stained for <i>Fgf8</i> expression in the second heart field. As shown by <i>in situ</i> hybridization for <i>Fgf8</i> transcripts, the e8.5 control embryo (A) displays numerous pharyngeal mesodermal cells exhibiting <i>Fgf8</i> expression (brown staining; black arrowheads). These cells represent progenitors of the right ventricle (RV) and outflow tract (OFT) that reside dorsal to the heart. <i>Fgf8</i> expression is also detected in the lateral pharyngeal endoderm (En) and pharyngeal ectoderm (Ec). In stark contrast, The <i>Gng5^−/−</i> mutants (B,C) have fewer pharyngeal mesoderm cells and reduced <i>Fgf8</i> expression in this region. The red arrow (B) indicates the most rostral portion of heart tube and the absence of OFT; the black arrowhead denotes absent <i>Fgf8</i> expression in the thin layer of pharyngeal mesoderm dorsal to the heart. In a more caudal section (C), the heart tube (HT) is visible and faint <i>Fgf8</i> expression is detected in the mesoderm adjacent to the heart tube (black arrowhead) and in the most proximal portion of the left side of the heart tube and adjacent mesoderm (red arrowhead). HF, head fold, P, pharynx; En, endoderm; Ec, ectoderm. <b>Panel D</b>, relative expression of proliferative markers in pharyngeal region of e9.0 control and <i>Gng5</i>^−/− mutant embryos. As shown by qPCR analysis, <i>Gng5^−/−</i> mutant embryos display reduced expression of three proliferative markers in the second heart field (* <i>p</i><0.002; ** <i>p</i><0.0001 by Student’s <i>t</i>-test), using elongation factor 1 (<i>Eef1a1)</i> as the housekeeping gene. All primer sequences can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090970#pone.0090970.s003" target="_blank">Table S1</a>.