Diverse expression patterns of LIM-homeodomain transcription factors (LIM-HDs) in mammalian inner ear development

Abstract

LIM-homeodomain transcription factors (LIM-HDs) are essential in tissue patterning and differentiation. But their expression patterns in the inner ear are largely unknown. Here we report on a study of twelve LIM-HDs, by their tempo-spatial patterns that imply distinct yet overlapping roles, in the developing mouse inner ear. Expression of Lmx1a and Isl1 begins in the otocyst stage, with Lmx1a exclusively in the non-sensory and Isl1 in the prosensory epithelia. The second wave of expression at E12.5 includes Lhx3, 5, 9, Isl2, and Lmx1b in the differentiating sensory epithelia with cellular specificities. With the exception of Lmx1a and Lhx3, all LIM-HDs are expressed in ganglion neurons. Expression of multiple LIM-HDs within a cell type suggests their redundant function.

Figure. 1

Expression of LIM-HDs in the developing inner ear: E10.5 (A,B), E12.5 (C-H) and E14.5 (I-O). Each stage was labeled with sidebars. A: ISL1 labeling in the region of future sensory epithelia (bracket) was distinct from primarily non-sensory PAX2 expression (red) in the medial portion of otocyst (Ot). OG, otic ganglions. B: In adjacent section, prominent Lmx1a expression was in a region overlapping with PAX2 expression. C: At E12.5, ISL1 expression was prominent in primordial cochlea (coch), cochleovestibular ganglions (CVG) and the developing saccular sensory epithelia, which overlapped slightly with PAX2 expression (arrow). D: Labeling with antibodies against LHX3 and POU4F3 showed immunoreactivities in nascent utricular hair cells. Most hair cells were LHX3 and POU4F3 double-positive (arrows to show example), whereas occasional early hair cells were only positive for POU4F3 (arrowhead). E-H: Expression of Lhx5, Lhx9, Lmx1a and Lmx1b in the inner ear at E12.5. Among them, only Lhx5 was expressed in the primordial cochlea (Fig.1E). Lmx1a was detected in the non-sensory epithelial region, but undetectable in the sensory epithelium positive for ISL1 (arrows, comparing to 1C) or in the cochleovestibular ganglions (Fig.1G). The sensory epithelial region of saccule is indicated by brackets, which correspond with ISL1 staining in adjacent section (data not shown). I: ISL1 expression in utricle at E14.5 was mainly in supporting cells (SC), with little expression in hair cells (HC). J: ISL2 expression was higher in saccular hair cells, weak in supporting cells and stroma. K: LHX3 expression was only in the utricular hair cells, coinciding with Myosin VIIa (MYO7A). L, M and O: Lhx5, Lhx9 and Lmx1b were detected in the sensory epithelial cell region. N: Robust Lmx1a expression was restricted to non-sensory epithelia including endolymphatic duct (ED), and was excluded in the vestibular ganglions (VG). Ut, utricle; Sac, saccule; Cri, crista; SE, sensory epithelia. Scale bar=20μm.

Figure 2

Expression of LIM-HDs in E16.5 vestibular system (A-H) and cochlea (I-P). A: ISL1 expression was mainly in utricular supporting cells. B: ISL2 expression was similar to E14.5, slightly stronger in hair cells than in supporting cells or stroma. C: LHX3 expression was upregulated in hair cells. D: In utricle, Lhx5 expression was in two regions associated with hair cells (brackets near the lumen), which was separated by a region with supporting cell expression (bracket close to the basal lamina). E, F and H: Lhx6, 9 and Lmx1b showed expression primarily in hair cell region. G: Lmx1a expression was greatly reduced and restricted to the transitional epithelium and dark cells of utricle and crista (brackets). I: ISL1 was expressed in all cochlear epithelial cells including hair cells and spiral ganglions (SG). J: ISL2 was widely expressed in the cochlea, with the labeling more prominent in hair cells. K: LHX3 was specifically expressed in hair cells. L,M,N,P: Lhx5, 6, 9 and Lmx1b were weakly expressed in cochlear hair cells, GER and SG. O: Lmx1a was expressed in the non-sensory epithelia (NSE), including primordial spiral prominence, stria vascularis and Reissner's membrane, as demarcated by the bracket. Scale bar=20μm.

Figure 3

Expression of LIM-HDs in E18.5 cochlea (A-H) and P6 inner ear (I-P). Each stage was labeled with sidebars. A: ISL1 was down-regulated in hair cells at E18.5, while was maintained in all other cochlear cells. B: ISL2 showed the same expression pattern as in E16.5, with slightly higher expression in hair cells than in other cochlear cells. C: Isl2 in situ hybridization showed the same pattern as shown with immunostaining, including spiral ganglions (SG). D: LHX3 showed the same hair cell expression pattern as in E16.5. E, F and H: Expression of Lhx6, Lhx9 and Lmx1b was similar to their respective expression patterns in E16.5. G: Expression of Lmx1a was maintained in the spiral prominence (SP, arrow) and down-regulated in the marginal cells (MC, arrowhead). I: At P6, ISL1 expression was completely absent in hair cells and most supporting cells, while it was maintained in Hensen cells (HeC) and GER. J: Isl1 in situ hybridization showed the result that matched the immunostaining in 3I with the exception in Hensen cells, which may indicate that the immunostaining had higher sensitivity. K: ISL2 expression was up-regulated in hair cells while was maintained at lower level in supporting cells and GER. L: Isl2 in situ hybridization showed expression patterns nearly identical to that obtained from immunostaining study (3K). M,O: Expression of Lhx3 and 9 in P6 utricle and cochlea. N: Lhx5 expression was in the P6 cochlear hair cells, spiral ganglion neurons and very weakly in the GER. P: Lmx1a expression was significantly down-regulated in the transitional epithelium of utricle (bracket). Scale bar=20μm.