Expression of Islet1 marks the sensory and neuronal lineages in the mammalian inner ear

Abstract

Several basic helix-loop-helix (bHLH) genes have been shown to be essential for the generation of the auditory sensory hair cells or the spiral ganglion (SG) neurons that innervate the hair cells in the cochlea, as well as a variety of cell types in the other nervous systems. However, it remains elusive what cellular context-dependent mechanisms confer the inner ear-specific neuronal or sensory competency/identities. We explored the possibility that one of the mechanisms responsible for generating cellular diversity in the nervous system through cooperative action of bHLH and LIM-homeodomain (LIM-HD) transcriptional factors might also contribute to the inner ear-specific sensory and/or neuronal competency. Here, we show that Islet1 (Isl1), a LIM-HD protein, is expressed early in the otocyst in the region that gives rise to both the auditory sensory organ, the organ of Corti, and SG neurons. Subsequently, the expression of Isl1 is maintained in SG neurons but is transitory in the sensory lineage. At embryonic day 12 (E12) in mice, the expression of Isl1 marks distinctively the ventral portion of the nascent cochlear epithelium encompassing the primordial organ of Corti. At E13, Isl1 is maintained at relatively high levels in the sensory primordium while down-regulated in the other regions of the cochlear duct. As the sensory epithelium starts to differentiate, it is down-regulated in the entire cochlear epithelium. The expression of Isl1 in the developing inner ear reveals an early and likely a common step in the development of both sensory and neuronal lineages of the inner ear, and suggests its potential role in the inner ear-specific sensory and neuronal cell development.

Fig. 1

Detection of Islet1 (Isl1) transcripts in the developing inner ear. A: The distinct stages of the developing mouse inner ear are illustrated, including the initial recognition of the placode, neurogenesis, and cochlea specification and growth. B: Isl1 was detected at higher levels at embryonic day (E) 13.5 than at postnatal day (P)1, compared with comparable expression of a house-keeping gene L19 at both stages.

Fig. 2

Expression of Islet1 (Isl1) in the inner ear neuronal lineage from embryonic day (E) 8.5 to E9.5. A–L: A vertical section from E8.5 (A–D) and a transverse ventral section from E9.5 (E–L) embryos were triply stained with antibodies against Pax2 (A,E,I, magenta), NeuroD (B,F,J, green), and Isl1 (C,G,K, red). D,H,L: Overlays of NeuroD and Isl1 staining. I,J,K,L: Higher magnified images of E,F,G,H, respectively. For E8.5 vertical section, dorsal is at the top, and for E9.5 transverse section, anterior is at the top. Medial is at the left for both the sections. VCG, vestibular–cochlear ganglion neurons. Scale bars = 50 μm in A (applies to A–D), E (applies to E–H), I (applies to I–L).

Fig. 3

Expression of Islet1 (Isl1) in the inner ear neuronal lineage at embryonic day (E) 10.5. A–D: Adjacent vertical (A,B) or transverse (C,D) E10.5 sections were stained for Isl1 (A,C) or Tuj-1 (C,D). Arrows in A and C delimit the expression domain of Isl1 in the otic epithelium at this stage. E–H: Transverse (E,F) and vertical (G,H) sections were triply labeled for Pax2 (E,G, magenta), Isl1 (F,H, red), and NeuroD (F,H, green). F,H: The staining for Isl1 and NeuroD was overlaid. VCG, vestibular–cochlear ganglion neurons. Scale bars = 50 μm in A (applies to A,B), C (applies to C,D), E (applies to E,F), G (applies to G,H).

Fig. 4

Islet1 (Isl1) expression in the vestibular-cochlear ganglion neurons and in the otic epithelium at embryonic day (E) 11.5. A–D: Adjacent otocyst sections at E11.5 (A,B and C,D) were stained with an antibody against Isl1 (Isl1, A,C) or neuronal β-tubulin (Tuj1, B,D). C,D: Higher magnifications of the ventral region in A and B, respectively. The green signal in A–D is from green fluorescent protein (GFP) expressed under the control of Math1 enhancers (Chen et al., 2002). Arrows in A and C indicate the boundaries of the Isl1-expressing ventral–medial region of the otocyst. Arrowheads in C and D indicate cells expressing the nucleus-localized GFP under the control of Math1 enhancers within the otocyst epithelium. AE,F: A transverse section triply stained for Isl1 (E,F, red), NeuroD (E,F, green), and Pax2 (E,F, magenta). E: The staining of Isl1 and NeuroD was overlaid to indicate specific staining for each (red for Isl1 and green for NeuroD) or for double labeling (yellow). F: All three labels were overlaid to indicate the relative expression domains of each gene. Note that the magenta colored Pax2 staining covers over other staining. Only complementary staining to Pax2 staining of Isl1 and NeuroD can be showed in F. Arrows in E,F indicate the Isl1-expressing domain in the otic epithelium that is negative for NeuroD staining. VCG, vestibular–cochlear ganglion neurons. Scale bars = 50 μm A (applies to A,B), C (applies to C,D), E (applies to E,F).

Fig. 5

Islet1 (Isl1) expression in the nascent vestibular sensory organs. A–D: Adjacent sections for the utricle were stained for a hair cell marker myosin VIIa (A), Isl1 (B), neuronal β-tubulin (D, Tuj1), or double stained for myosin VIIa (C, red, cytoplasmic staining) and Isl1 (C, green, nuclear staining). Arrowheads in B,C indicate Isl1 and myosin VIIa double-positive cells. Scale bar = 50 μm in A (applies to A–D).

Fig. 6

Islet1 (Isl1) expression in the cochlea. A–I: Adjacent cochlear sections from embryonic day (E) 12.5 (A–C), E13.5 (D–F), and E14.5 (G–I) stained for Isl1 antibody (A,D,G), Isl1 mRNA (B,E,H), neuronal β-tubulin antibody (C, Tuj1), p27^Kip1 antibody (F), or with Math1/GFP signal (I). Arrowheads in A–C mark the boundaries of the ventral cochlear epithelium expressing Isl1 at E12.5. Brackets (D–I) indicate the primordial organ of Corti before terminal differentiation. SG, spiral ganglion neurons. Medial is at the left of the images. GFP, green fluorescent protein. Scale bar = 50 μm in A (applies to A–I).

Fig. 7

Differential expression of Islet1 (Isl1) in the sensory and neuronal cells of the inner ear. A–D: Cross-sections of the cochlea (A), saccule (B), utricle (C), and crista (D) at embryonic day 16.5 double stained for Isl1 (A, green) and Math1/GFP (A, red) antibodies, or Isl1 (B–D, green) and myosin VIIa (B–D, red) antibodies. The brackets indicate the organ of Corti in the cochlea (A), or the hair cell layer in the vestibular sensory organs (B–D). The arrows indicate the supporting cell layer in the vestibular sensory organs (B–D). Isl1 is absent in the sensory organs but persists in SG neurons. SG, spiral ganglion neurons; GFP, green fluorescent protein. Scale bar = 50 μm in A (applies to A–D).