Expression of NR2F2 in the embryonic rat cochlea.

<p><i>a</i>. Overview of NR2F2 expression throughout the cochlea at E18. NR2F2 is most strongly expressed in the cochlear duct. In the apical duct, strong expression is seen in the super-medial wall (<i>S-MW</i>), greater epithelial ridge (<i>GER</i>), and lesser epithelial ridge (<i>LER</i>). This expression is reduced to the lesser epithelial ridge in the basal duct (<i>arrowhead</i>). <i>b–e</i>. Apical cochlear duct; <i>f–i</i>. Middle cochlear duct; and <i>j–m</i>. Basal cochlear duct. <i>b, f & j</i>, cell nuclei stained with DAPI; <i>c, g & k</i>, NR2F2 labelling; <i>d, h & l</i>, filamentous actin labelled by Phalloidin; and <i>e, i & m,</i> Merge of DAPI (<i>blue</i>), NR2F2 (<i>green</i>) and Phalloidin (<i>red</i>), s<i>cale bars</i>: 100 µm in <i>a</i> and 20 µm in <i>e, i & m</i>.</p

POU4F3-mediated activation of PRE1, PRE2 and a 4.2 kb <i>Nr2f2</i> 5′ flanking sequence.

<p><i>a</i>. Schematic diagram of reporter constructs used in luciferase reporter assays in ND7 cells. POU4F3 recognition element (PRE) 1 and 2 are shown in an <i>Nr2f2</i> 5′ flanking region fragment (<i>grey box</i>) cloned upstream of a luciferase reporter gene (<i>LUC</i>). The location of this fragment relative to the <i>Nr2f2</i> transcriptional start site is shown. <i>b</i>. Evaluation of the response of 4.2 kb-<i>Nr2f2</i>-Luc to increasing levels of POU4F3 or <i>dreidel</i> mutant POU4F3 (<i>Ddl</i>) expression construct. The luciferase activity of the reporter is normalised to its response to the empty expression vector and results are expressed relative to this. <i>c</i>. Response of the PRE1-Luc reporter construct in co-transfection experiments with POU4F3 and <i>dreidel</i> expression constructs. <i>d</i>. Evaluation of the response of PRE2-Luc in similar experiments to <i>c</i>. Error bars represent the s.e.m in <i>b, c</i> and <i>d</i> (n = 6 for each data point).</p

POU4F3 recognition element mutation attenuates POU4F3 binding and activation of the <i>Nr2f2</i> 5′ flanking region.

<p><i>a</i>. EMSA analysis of POU4F3 recognition element mutations on POU4F3 binding. Probes were either incubated alone or with <i>in vitro</i> translated POU4F3. <i>Closed arrowheads</i> indicate POU4F3-specific PRE1 bandshifts and the <i>open arrowhead</i> indicates the POU4F3-specific PRE2 bandshift. The mutation of each site severely compromises the ability of POU4F3 to bind to each sequence. <i>b</i>. Luciferase reporter assay upon co-transfection of POU4F3 with the 4.2 kb-<i>Nr2f2</i>-Luc-Mut reporter construct. Error bars represent the s.e.m (n = 6 for each data point).</p

Identification and verification of POU4F3 recognition elements in the <i>Nr2f2</i> 5′ flanking region.

<p><i>a</i>. Schematic diagram of the location of two putative POU4F3 recognition elements (PREs) identified in the <i>Nr2f2</i> 5′ flanking region and used in EMSA analysis. Underlined bases correspond to POU4F3 binding sites predicted by MatInspector software with capital letters denoting matches to the core sequence. The asterisks signify matrix position conservation >60/100 (<i>TSS</i>, transcriptional start site and <i>UTR</i>, un-translated region). <i>b</i>. Sequences shown in <i>a</i> were used as radiolabelled probes in EMSA analysis with <i>in vitro</i> translated POU4F3 or a Luciferase control. Bandshifts due to protein-specific binding by POU4F3 are indicated by arrowheads. <i>c</i>. The same probes were used in EMSA analysis with UB/OC-2 cell nuclear protein extract. Reactions were incubated either alone, with UB/OC-2 cell nuclear protein extract or with the nuclear protein extract and an excess of non-radiolabelled competitor oligonucleotide as indicated. The ‘<i>P</i>’ suffix refers to non-radiolabelled POU4F3 binding sequence whereas the ‘<i>A’</i> suffix indicates a non-radiolabelled AP4 binding sequence. POU4F3-sequence-specific shifts are indicated by arrowheads.</p

Evolutionary conservation of transcription factor binding sites in the <i>Nr2f2</i> 5′ flanking region.

<p>Schematic representation of the conservation of the region surrounding the human PRE1 sequence (<i>a</i>) and the human PRE2 sequence (<i>b</i>) compared to corresponding mouse, rat and chimpanzee sequences. <i>a</i>. The well-conserved PRE1 sequence is indicated by asterisks with evolutionarily conserved binding sites for the POU domain transcription factors OCT1 and OCT_C indicated by horizontal bars. Conserved bases are represented by black boxes with mismatches in grey. <i>b</i>. PRE2 is less well conserved with eight mismatched bases and no corresponding POU domain transcription factor binding sites in the TRANSFAC database. Conserved bases are again represented by black boxes with mismatches in grey.</p

Expression of NR2F2 in the postnatal mouse inner ear.

<p>Cryotome sections of P1 and adult mouse inner ears were subjected to immunohistochemistry in order to characterise their postnatal NR2F2 expression pattern. NR2F2 labelling is seen throughout the sensory epithelia of the cochlea and cristae. In the cochlea, NR2F2 labelling is of greater intensity in the apex than the base. NR2F2 is expressed in the nuclei of apical hair cells at all ages examined. In P1 mice, NR2F2 expression is reduced in basal hair cells. In adult mice, the expression of NR2F2 in basal hair cell nuclei appears further reduced. NR2F2 is also expressed in the hair cells of the cristae of the semicircular canals and this expression is maintained into adulthood. In merged images DAPI is <i>blue</i>, NR2F2 is <i>green</i>, Phalloidin is r<i>ed</i>, a<i>rrowheads</i> indicate hair cells and s<i>cale bars</i>: 20 µm.</p