Figure 4. Expression analysis of Hmx3 and Hmx2 homeobox transcription factors in hb/hb and control littermates.

A–B: Hmx3 expression in hb/hb mutants tested by whole mount RNA in situ hybridisation at E10.5, showing decreased expression of Hmx3 in the dorsal part of the otocyst of hb/hb mutants compared to littermate controls (arrows) C,D: RNA in situ hybridisation for Hmx3 at E12.5 in vestibular system sagittal sections. In control littermates, Hmx3 RNA is detected in the canal fusion plate (black arrowhead in C), semicircular canals (red arrowheads in C), and in the utricle and saccule (not shown) but its expression is always observed in non-sensory epithelial cells as previously reported (C). In hb/hb mutants we still detect Hmx3 mRNA in the vestibular non-sensory regions compared to the littermate controls (arrowhead in D). E,F: Hmx2 expression in vestibular system detected by in situ hybridisation on sagittal sections from hb/hb and littermate controls at E12.5. In control mice, as previously reported, Hmx2 shows a similar expression pattern to Hmx3 in the non-sensory cells and in the canal plate, in the utricle (arrow in E) and in the canals (not shown). In hb/hb we detect Hmx2 expression only in a few cells in the non-sensory regions of the structurally abnormal vestibular system, compared to the littermate controls (arrowheads in F). Scale bars: A,B, 0.5 mm; C–F, 100 µm. G: Quantitative real-time PCR of cDNA generated from RNA from E12.5 littermate embryo half heads. Only Hmx3, Hmx2 and Nkx1.2 mRNA levels are significantly downregulated in hb/hb compared to littermate controls. Error bars, s.d. Quantity normalised to Hprt1 levels. N = 3. *:p<0.05; **: p<0.01. H–I: Expression of Hmx3 in hb/hb cochlear sections and control littermates at postnatal day five. After birth, Hmx3 is expressed in intermediate cells in stria vascularis (arrowhead in H, see also Figure S2). As it is clear in I, no Hmx3 staining is detected in hb/hb cochlea at P5, consistent with the loss of intermediate cells (arrowhead in I). Scale bars: 20 µm. a: anterior, asc: anterior semicircular canal, cr:cristae, cy: vestibular cyst, D: distal; ed: endolymphatic duct fm: fused maculae lsc: lateral semicircular canal mu: maculae utriculi ms: maculae sacculi; oc: organ of corti, ov: otic vescicle; rm: Reissner's membrane, s: saccule scp: superior canal plate; sd: semicircular duct; s+u: utriculosaccular space, sv: stria vascularis, vd: vestibular diverticulum.