The generation of the inner ear

Generation o f the inner ear involves a wide range o f cellular phenotypes whose formation implies a series o f cel I-fate decisions during otocyst differentiation. In addition, several phenotypes are segregated into specialized areas in a highly organized fashion. What factors and how they contribute to the complicated formation is still largely unknown. During the past few years a large number o f genes expressed during the early stages o f inner ear development have been identified (for review see Fekete 1996; Torres and Giraldez, 1998). The expression o f these genes has been speculated to be associated with the morphogenesis o f structures such as the semicircular canals (e.g. Nkx-5.1), or sensory organs (bone morphogenic protein 4, BMP-4) (see below). The collective expression of some o f these markers were suggested to map the otic vesicle and provide reference to the morphogenesis or designation o f cell types (Feteke, 1996, see following). Although the proposed explanation still lacks sound support, it is now generally agreed that heterogeneity

already exists before overt differentiation occurs during the otic pit and otic vesicle stage, be it o f axis determination or cell specification. The following section will discuss the known facts about the gross morphological changes, regulation o f otic induction, and the mechanisms o f regional specification, followed by a discussion o f how different cell types differentiate in the inner ear.

1.15.3.1 Early morphological changes

The first identifiable stage o f inner ear development begins with the formation o f the otic placode from the surface ectoderm o f the embryo. It can already be recognized in apposition to the neural tube in the 3-6 somite embryo (reviewed by Torres and Giraldez, 1998). The otic placode then invaginates to form the otic cup and then the otic vesicle (Anniko, 1983; reviewed by Feteke, 1996; Torres and Giraldez, 1998). Neuroblasts have been seen delaminating from the otic cup o f the zebrafish embryo to form the cochleovestibular ganglion (Haddon and Lewis, 1996). It is not directly proved but the generation of cochlear and vestibular ganglion in mammals is believed to be similar (Torres and Geraldez, 1998). As development proceeds this single ganglion then splits into the cochlear ganglion and vestibular ganglion (reviewed by Torres and Geraldez). The endolympatic duct is the first among the structures to elongate from the oval shaped vesicle as it matures, E4 (stage22) in the chick (Bissonnette and Fekete, 1996) and E l 2 o f rat (Wu and Oh, 1996; MJ Lee, unpublished). This is followed by the elongation o f the cochlear duct at E5 (stage24) o f chick (Lillie, 1952; Bissonnette and Fekete, 1996), and E l 3 o f rat (M-J Lee, unpublished). The pouch-like anlagen o f the semicircular canals develops into its final tubular morphology very quickly during E6 o f chick (Bissonnette and Fekete,

1996). The specialization o f distinct structures is believed to happen before distinct morphological structures can be identified (see below). Morphologically distinct hair cells and supporting cells do not appear until E l 7-18 in rat, or E l 3-14 o f mouse (Ryan, 1997).

1.15.3.2 Regional specification

The determination o f the polarity o f the otic vesicle takes place early, soon after the induction o f the otic placode. Different CAMs, which are believed to expressed in

specific modal patterns, are differentially expressed in otic placode as early as the beginning o f the induction (Richardson et al, 1987). Based on the pattern o f gene expression o f several pattern formation related genes, Fekete proposed a boundary theory for regional specification (see above). The model states that at the otic pit and/or otic vesicle stage, the vesicle is subdivided by boundaries into compartments. The boundaries are established at the limits o f gene expression domains, or by other secreted inductive factors from adjacent tissue, i.e. the neural tube, the notochord, neighbouring ectodermal tissue (Feteke, 1996; Lawrence and Struhl, 1996). The nature o f the signal proposed for the latter probably belongs to the same family o f secreted factors which determine the dorsal-ventral axis o f neural tube, the antennapedia class homeobox genes or other transcription factors that are known to regulate the segmentation o f the hindbrain. Three boundaries (mediolateral, anterioposterior, and dorsoventral) and six points o f intersection can be identified this way. Sensory-competent cells will become specified to form sensory organs only at the point o f intersection or adjacent to boundaries. This supposedly not only specifies the location o f the organ but also the type o f the organ (Fekete, 1996).

Before distinct morphological structures are observed, expression o f some early genes separates into patches and later in development corresponds well to specific sensory structures. Based on this type o f observation several genes expressed during early otic development were suggested to be markers for the primordium o f certain structures. BMP-4 is suggested to be a marker for the sensory organs, based on the good association o f its expression in the gradually recognizable sensory primordium in the chick. p75NGF-R and BDNF are expressed in different patterns on cristae and maculae and can be used to distinguish these two structures (Wu and Oh, 1996). The BMP-4 gene is expressed in the rat inner ear as well (chaper 5). Nkx-5.1 is suggested to be a marker for the semicircular canals in the mouse. It is expressed in the otic vesicle in a restricted dorsal-lateral pattern, and the expression persists in the vestibular structures throughout inner ear development. Mice with a null mutation o f Nkx-5.1 fail to develop semicircular canals (Rinkwitz-Brandt et al., 1996; Hadrys et al., 1998). Pax-2 is suggested to be marker for the cochlear duct in the mouse. Apart from these murine genes, in the chick GH-5 and SOHo-1 are suggested to be markers

o f semi-circular canals, including both sensory and non-sensory parts o f the canals (Fekete, 1996).In zebrafish mshC, a member o f the homeobox gene family, is expressed only in the maculae (Ekker et al., 1992).

1.15.3.3 The specification of sensory organs versus specification o f other structures

Interestingly, the specification o f polarity o f the gross anatomy and the specification o f the sensory organ seems to be regulated by different mechanisms, as suggested by the fact that in some mutations when there is severe deformation o f the inner ear morphology, sensory organs with distinct hair cells and supporting cells are still present, as in the case when otic epithelium (without surrounding mesenchyme) is transplanted to a non-otic field (Swanson et al., 1990) , or in mice lacking Hoxa-1 or FGF-3, and in kreisler mutant mice (Mark et al., 1993; Chisaka et al., 1992; Mansour et al., 1993; McKay et al., 1996). Early expression o f cristae markers suggests that their specification proceeds independently o f the canals in which they normally reside. This idea is further supported by the observation that cristae markers are evident even in the abnormal or failed semicircular canal formation in zebrafish mutants eselsohr and znikam (Whitfield et al.,1996; Malicki et al., 1996). In experiments when the otic vesicle in the chick was rotated 180° around its vertical axis in situ, the vesicle expressed Pax-2 in a pattern corresponding to the new surrounding tissue, suggesting that the Pax-2 expression is not determined at the stage o f vesicle formation (Linberg et al., 1995).

Recent experiments suggest that the specification o f sensory epithelium might occur earlier than the specification o f morphological structures. In transplant experiments o f otocyst o f E2.5 chick, it was shown that whereas the anterior-posterior (A/P) axis o f the sensory organs is fixed at the time o f transplantation, the A/P axis for most non­ sensory structures, is fixed later (Wu et al., 1998). Different compartments o f the inner ear seem to be regulated independently during morphogenesis. This could be demonstrated in the Pax-2 and Nkx-5.1 null mutations. As mentioned above, the Pax- 2 gene is related to the development o f cochlear duct while Nkx-5.1 is important in the forming o f semicircular canals. Up to mouse ElO the gene expression o f Pax-2 is

not affected in the Nkx-5.1 knock out mice, nor vice versa, suggesting that the gene which is responsible for defects in forming the semicircular canals is not involved in the formation o f the cochlear duct, and vice versa (Hadrys et al., 1998; Torres and Giraldez, 1988). However, a clever mechanism works to impose the framework o f morphogenesis on the future sensory area. BMP-4 is expressed at the otic cup stage in two restricted patches beside the neurogenic area. During later development some sensory areas originate by segregation from an initially continuous future sensory area, while others appear de novo, judging from the BMP-4 expression. The fact that cristae and maculae o f different structures could be derived from a common sensory patch suggests an additional mechanism might exist to correlate morphogenesis and sensory induction (Wu and Oh, 1996; Torres and Giraldez, 1988). Although the mechanism regulating the main specification events remains elusive, there is now consensus that the axis specification and sensory organ formation involves multi-step regulation.

1.15.4 Cell lineages of the inner ear