Expression of Prox1 during mouse cochlear development

Abstract

We carried out an analysis of the expression of Prox1, a homeo-domain transcription factor, during mouse inner ear development with particular emphasis on the auditory system. Prox1 is expressed in the otocyst beginning at embryonic day (E)11, in the developing vestibular sensory patches. Expression is down regulated in maturing (myosin VIIA immunoreactive) vestibular hair cells and subsequently in the underlying support cell layer by E16.5. In the auditory sensory epithelium, Prox1 is initially expressed at embryonic day 14.5 in a narrow stripe of cells at the base of the cochlea. This stripe encompasses the full thickness of the sensory epithelium, including developing hair cells and support cells. Over the next several days the stripe of expression extends to the apex, and as the sensory epithelium differentiates Prox1 becomes restricted to a subset of support cells. Double labeling for Prox1 and cell-type-specific markers revealed that the outer hair cells transiently express Prox1. After E18, Prox1 protein is no longer detectable in hair cells, but it continues to be expressed in support cells for the rest of embryogenesis and into the second postnatal week. During this time, Prox1 is not expressed in all support cell types in the organ of Corti, but is restricted to developing Deiters' and pillar cells. The expression is maintained in these cells into the second week of postnatal life, at which time Prox1 is dynamically down regulated. These studies form a baseline from which we can analyze the role of Prox1 in vertebrate sensory development.

Figure 1

Section through the otocyst at E11 labeled with Prox1 antibody (red). The arrows point to three regions of Prox1 immunoreactivity. R = rostral, M = medial. Scale bar = 50μm

Figure 2

In situ hybridization with a probe to mProx1. (A) A section through the otocyst at E14 showing the developing vestibular structures which all express Prox1, arrow points to developing utricle and arrowheads to developing cristae of the semicircular canals. (B) A section of an E16 cochlea, arrowheads indicate the developing organ of Corti and arrows the spiral ganglion. (C, D, E) Higher magnification images of the staining in the developing organ of Corti. There is more intense labeling in the base (C, D) relative to the apex (E) at this stage in development. The scale bar in A = 50 μm and applies also to B and the scale bar in D = 20 μm and applies to C-E.

Figure 3

Sections through the otocyst of an E14 embryo showing Prox1 (red) and Myosin VIIA (green) immunoreactivity in the developing vestibular organs. (A) A section through the developing saccule. (B) A section through the developing utricle. (C) A crista from one of the semicircular canals. (D) A section through the developing saccule that shows some double-labeled hair cells (arrowheads). Scale bar = 50 μm

Figure 4

Sections through the cochlea of an E14 mouse labeled with antibodies against p27 (A, C), Myosin VIIA and Prox1 (B, D). The area in the box in A is shown at higher magnification in C, while the area in the box in B is shown at higher magnification in D. The arrowheads in A and C point out the zone of non-proliferating cells (ZNPC) expressing p27. The arrow in B points out the spiral ganglion, SG, which shows Prox1 immunoreactivity at this age. (D) shows the lack of labeling for either Prox1 or Myosin VIIA at E14 (arrowhead). In panels A and B the apex is located at the top of the figure. Scale bar = 100μm

Figure 5

Sections through the cochlea of E15 (A, B, C) and E14.5 mouse embryos labeled with antibodies against p27 (A), Myosin VIIA and Prox1 (B, C), and Prox1 (D). (A) The ZNPCs can be clearly identified with p27 labeling (arrowheads). (B) An adjacent section showing the Myosin VIIA and Prox1 immunoreactivity. The cells of the SG are strongly labeled for Prox1, while a subset of cells within the ZNPC at the basal end of the cochlea label for both Myosin VIIA and Prox1. (C) The double-labeling in the region in the box in B is shown at higher magnification. The arrow points out the Myosin VIIA (green) labeled hair cells, while immediately adjacent to them are Prox1 labeled nuclei (red). (D) Prox1 immunoreactive cells at E14.5, at the base of the cochlea (arrow). In panels A and B, the apex is located at the top of the figure. Scale bar = 100μm in A and B and 25μm in C and D.

Figure 6

Sections through the cochlea of E14.5 and E15.5 mouse embryos that express GFP under the control of the Math1 promoter. (A) The Math1-GFP labeled inner hair cells appear at E14.5 (green), and are located immediately adjacent to the Prox1 immunoreactive cells (red) at this stage. (B) The Math1-GFP hair cells are now present in both the LER and the GER at E15.5; Prox1 immunoreactive cells are present primarily in the support cell layer of the LER, though several Math1 expressing hair cells are also immunoreactive for Prox1. (C-E) Higher magnification of the organ of Corti shown in B, to better show the double-labeling (arrow) of Prox1 (E) and GFP (C). LER: lesser epithelial ridge, GER: greater epithelial ridge. Scale bar = 20 μm.

Figure 7

Sections through the cochlea of E16 mouse embryos showing labeling of p27 (A, D), Myosin VIIA and Prox1 (B, C). The micrograph shown in C is a higher magnification view of the region in the box in B. The section shown in B is an adjacent section to that shown in A. (B) The spiral ganglion (SG) cells express Prox1 at this stage, as do many of the cells in the LER. (C) The inner hair cells (green, arrowhead) express Myosin VIIA at this stage, but are not labeled with the Prox1 antibody (red). (D) A section of E16 mouse cochlea showing that only a subset of the p27 positive cells (red) also label for Prox1 (green). LER: lesser epithelial ridge, GER: greater epithelial ridge. Scale bar = 100 μm in B and 20 μm in D.

Figure 8

Sections through the cochlea of E18 mouse embryos showing labeling of Myosin VIIA (green) and Prox1 (red). (A) Low power micrograph showing the entire cochlea. (B-D) Higher magnification views of the regions indicated by the arrows in A. Prox1 labels the nuclei of the Deiters’ and pillar cells but not the inner phalangeal cell (asterisks). Scale bar = 100μm

Figure 9

Confocal micrograph of a section through the apical cochlea of an embryonic day 17 mouse labeled for S100A1 (red) and Prox1 (green). (A; B; C) Prox1, merged and S100A1 labeling, respectively. (D) Higher magnification view of B. Arrow points to the S100A1 labeling of the inner hair cell, while the arrowhead points to the pillar cell nuclei, which are positive for Prox1 and negative for S100A1. H: Hensen cell, DC: Deiters’ cell, PC: pillar cell, IHC: inner hair cell, OHC: outer hair cell, IPC: inner phalangeal cell and B: border cell. Scale bar = 10μm

Figure 10

Sections through the cochlea of postnatal day 0 mouse showing labeling of Myosin VIIA (green) and Prox1 (red). (A) Low power micrograph showing the entire cochlea. (B, C) Higher magnification views of the regions indicated by the boxes in A. Prox1 labels the nuclei of the Deiters’ and pillar cells but not the hair cells. SG = spiral ganglion. Scale bar = 100 μm

Figure 11

Confocal images of whole-mounted organ of Corti pieces from basal (A, D, G), middle (B, E, H), and apical (C, F, I) regions of the cochlea at P3 (A-C), P5 (D-F), and P10 (G-H). All panels show Prox1 immunolabeling in the support cell nuclear layer of the epithelium. Approximately 10 1-μm slices from Z series stacks were brightest-point projected to capture the support cell nuclei in each image. IPC (inner pillar cells), OPC (outer pillar cells), DC-1, DC-2, and DC-3 (first through third rows of Deiters’ cells). The arrowheads in E point to regions lacking Prox1 immunoreactivity in the first and second rows of Deiters’ cells and in the outer pillar cells. The arrow in F points to IPC nuclear labeling. Scale bar = 15μm.

Figure 12

Diagram showing the relationship between prospero (grey) expression and the development of the external sensory organ cells in Drosophila (top) and that of Prox1 (grey) and the developing cochlear cells (bottom). SOP = sensory organ precursor; SEP = hypothetical sensory epithelial precursor in the inner ear; GER = greater epithelial ridge; LER = lesser epithelial ridge.