Notch signaling in the development of the inner ear: lessons from Drosophila

Abstract

The sensory patches in the ear of a vertebrate can be compared with the mechanosensory bristles of a fly. This comparison has led to the discovery that lateral inhibition mediated by the Notch cell-cell signaling pathway, first characterized in Drosophila and crucial for bristle development, also has a key role in controlling the pattern of sensory hair cells and supporting cells in the ear. We review the arguments for considering the sensory patches of the vertebrate ear and bristles of the insect to be homologous structures, evolved from a common ancestral mechanosensory organ, and we examine more closely the role of Notch signaling in each system. Using viral vectors to misexpress components of the Notch pathway in the chick ear, we show that a simple lateral-inhibition model based on feedback regulation of the Notch ligand Delta is inadequate for the ear just as it is for the fly bristle. The Notch ligand Serrate1, expressed in supporting cells in the ear, is regulated by lateral induction, not lateral inhibition; commitment to become a hair cell is not simply controlled by levels of expression of the Notch ligands Delta1, Serrate1, and Serrate2 in the neighbors of the nascent hair cell; and at least one factor, Numb, capable of blocking reception of lateral inhibition is concentrated in hair cells. These findings reinforce the parallels between the vertebrate ear and the fly bristle and show how study of the insect system can help us understand the vertebrate.

Figure 1

(Left) An optical section (confocal image) in the plane of the chick basilar papilla, showing the mosaic of hair cells (stained with HCA antibody, white blobs) and supporting cells (outlined by their cortical actin, stained with fluorescent phalloidin). (Reproduced from ref. .) (Right) Lateral inhibition with feedback: a simple model of Delta-Notch signaling that can generate spatial pattern. Only two cells are shown, interacting to adopt two different fates, but the same principles apply to a field of many cells, which by interacting in this way can in theory give rise to the type of pattern seen at Left.

Figure 2

Blocking Notch signaling causes down-regulation of Ser1. (A–C) Infection with RCAS-Dl1^dn virus, blocking signaling via Notch. Three consecutive sections through the utricle of an 8-day (E8) embryo are shown. The expression of Dl1^dn is shown by in situ hybridization with a Dl1 probe (red) (A and C) and by antibody staining (green) (B). Ser1 expression is shown by antibody staining (green) (A and C); the distribution of hair cells is shown with the HCA antibody (detecting hair bundles, red) (B). Ser1 expression is lost at sites of infection. Presence of hair cells proves that these sites lie within a sensory patch, where normally Ser1 is expressed. (D) Infection with RCAS-XSu(H)^dn. Infection was detected by antibody against viral gag protein (red) and Ser1 expression by Ser1 antibody (green). Again, the two stains are mutually exclusive: cells infected within the sensory patch down-regulate Ser1.

Figure 3

Infections with RCAS-Dl1^dn virus. (A and B) Sections through utricle at E8; the Dl1^dn-expressing patches abut but do not overlap sensory patches. Two examples are shown. Dl1^dn expression is shown with an antibody against Dl1 (green), hair cells with HCA antibody (red). (C and D) Dl1^dn-expressing cells often end up as neurons in the cochleovestibular ganglion. (C) Section of basilar papilla and underlying ganglion at E6 stained by in situ hybridization for Dl1^dn (red) and with Islet1/2 antibody (green) to mark the nuclei of neurons in the cochleovestibular ganglion. (D) Adjacent section stained with Dl1 antibody, revealing the dendrites of infected neurons with Dl1^dn in their membranes.

Figure 4

Overexpression of Dl1 in a sensory patch does not inhibit hair-cell production. (A–D) Sections of utricle at E8, infected with RCAS-Dl1 virus. (A) Infected patch stained with an antibody against Dl1 (green) and with HCA antibody (red). (B) The same scene, showing the HCA signal only, to reveal the distribution of hair cells more clearly. (C) Adjacent section stained by in situ hybridization for Dl1 (red) and with Ser1 antibody. (D) Same scene as in C, with the red fluorescence hidden to show that Dl1, in contrast with Dl1^dn, does not cause down-regulation of Ser1. (E) Specimen infected with pseudotype (replication-defective) virus expressing Dl1 + GFP, and stained with antibody against GFP (green) plus HCA antibody (red). Hair cells are produced normally even within clusters of contiguous cells all expressing Dl1.

Figure 5

C-Numb expression during sensory patch development. (A) Section of otic epithelium at E3, stained with Numb antibody (green) and counterstained with the nuclear dye Syto16 (red); note basal localization of Numb, apparently in all cells, including those undergoing mitosis close to the lumen. (B) Section of basilar papilla at E12, stained with Numb antibody (green) and HCA (red); the hair cells preferentially contain Numb, and it is no longer basally localized.

Figure 6

Summary diagram of Notch-mediated interactions in a newly differentiated sensory patch in the chick ear. See Discussion for commentary.