Systematic assignment of fossil otoliths is virtually always based on studies of otolith morphology and subsequent comparisons with otoliths from collections and/or literature. Although this usually represents a practical method, comparisons and subsequent evaluation may be biased by subjective criteria used in the individual descriptions. Quantitative morphometric studies focusing on variations in the otolith morphology of extant fishes have been conducted in fisheries research, mostly based on Fourier shape analysis and related methods. However, with regard to fossil otoliths, these approaches are generally not suitable, mainly due to preservation-related problems. Here we present a new approach for quantifying otolith variation between species and populations of killifish (cyprinodontiforms) in the genera Aphanius Nardo and daggerProlebias Sauvage that can be used with both extant and fossil otoliths. Our new approach includes the definition of 10 variables from linear and angle measurements of an otolith and statistical analyses. Best results were obtained by presorting the otoliths into three groups based on sulcus shape (straight, bent, S-shaped). In this case, canonical discriminant analysis (CDA) with jackknifed cross-validation yielded an overall species classification success of 86-96%. The three groups based on sulcus shape separate according to zoogeographic patterns (i.e., Mediterranean Aphanius, Arabian Aphanius, European daggerProlebias) and probably reflect phylogenetic lineages. Application of CDA to compare otolith variation between populations resulted in an overall classification success (jackknifed) of 33-83%. High levels of variation were observed for Aphanius dispar and daggerProlebias malzi, but not for A. fasciatus and daggerP. weileri. We suggest that otolith variation between populations results predominantly from geographic separation. Combination of qualitative characters (sulcus morphology) with quantitative approaches (otolith morphometry) presents a new approach for obtaining a better understanding of the taxonomy, diversity, and zoogeography of both fossil and extant killifishes. Moreover, the method may also be suitable for assessing taxonomy and diversity in other species-rich groups like the atheriniforms and many perciforms because these groups display otolith Bauplans that are similar to those seen in killifishes.

The avian cochlear duct houses both a vestibular and the auditory sensory organ (the lagena macula and basilar papilla, respectively) that each have a distinct structure and function. Comparative mRNA in situ hybridization mappings conducted over the time course of chicken cochlear duct development reveal that Wnt-related gene expression is concomitant with various developmental processes such as regionalization, convergent extension of the cochlear duct, cell fate specification, synaptogenesis, and the establishment of planar cell polarity. Wnts mostly originate from nonsensory tissue domains while the sensory primordia preferentially transcribe Frizzled receptors, suggesting that paracrine Wnt signaling predominates in the cochlear duct. Superimposed over this is the strong expression of two secreted Frizzled-related Wnt inhibitors that tend to show complementary expression patterns. Frzb (SFRP3) is confined to the nonsensory cochlear duct and the lagena macula, whereas SFRP2 is maintained in the basilar papilla along with Fzd10 and Wnt7b. Flanking the basilar papilla are: Wnt7a, Wnt9a, Wnt11, and SFRP2 on the neural side; and Wnt5a, Wnt5b, Wnt7a on the abneural side. The lateral nonsensory cochlear duct continuously expresses Frzb and temporarily expresses Wnt6 and SFRP1. Characteristic for the entire lagena is the expression of Frzb; in the lagena macula are Fzd1, Fzd7, Wnt7b; and in the nonsensory tissues are Wnt4, Wnt5a. Auditory hair cells preferentially express Fzd2 and Fzd9, while the main receptors expressed in vestibular hair cells are Fzd1 and Fzd7, in addition to Fzd2 and Fzd9.

Vertebrate hearing organs manifest cellular asymmetries across the radial axis that underlie afferent versus efferent circuits between the inner ear and the brain. Therefore, understanding the molecular control of patterning across this axis has important functional implications. Radial axis patterning begins before the cells become postmitotic and is likely linked to the onset of asymmetric expression of secreted factors adjacent to the sensory primordium. This study explores one such asymmetrically expressed gene, Wnt9a, which becomes restricted to the neural edge of the avian auditory organ, the basilar papilla, by embryonic day 5 (E5). Radial patterning is disrupted when Wnt9a is overexpressed throughout the prosensory domain beginning on E3. Sexes were pooled for analysis and sex differences were not studied. Analysis of gene expression and afferent innervation on E6 suggests that ectopic Wnt9a expands the neural-side fate, possibly by re-specifying the abneural fate. RNA sequencing reveals quantitative changes, not only in Wnt-pathway genes, but also in genes involved in axon guidance and cytoskeletal remodeling. By E18, these early patterning effects are manifest as profound changes in cell fates [short hair cells (HCs) are missing], ribbon synapse numbers, outward ionic currents, and efferent innervation. These observations suggest that Wnt9a may be one of the molecules responsible for breaking symmetry across the radial axis of the avian auditory organ. Indirectly, Wnt9a can regulate the mature phenotype whereby afferent axons predominantly innervate neural-side tall HCs, resulting in more ribbon synapses per HC compared with abneural-side short HCs with few ribbons and large efferent synapses.

The function of the inner ear critically depends on mechanoelectrically transducing hair cells and their afferent and efferent innervation. The first part of this review presents data on the evolution and development of polarized vertebrate hair cells that generate a sensitive axis for mechanical stimulation, an essential part of the function of hair cells. Beyond the cellular level, a coordinated alignment of polarized hair cells across a sensory epithelium, a phenomenon called planar cell polarity (PCP), is essential for the organ's function. The coordinated alignment of hair cells leads to hair cell orientation patterns that are characteristic of the different sensory epithelia of the vertebrate inner ear. Here, we review the developmental mechanisms that potentially generate molecular and morphological asymmetries necessary for the control of PCP. In the second part, this review concentrates on the evolution, development and function of the enigmatic efferent neurons terminating on hair cells. We present evidence suggestive of efferents being derived from motoneurons and synapsing predominantly onto a unique but ancient cholinergic receptor. A review of functional data shows that the plesiomorphic role of the efferent system likely was to globally shut down and protect the peripheral sensors, be they vestibular, lateral line or auditory hair cells, from desensitization and damage during situations of self-induced sensory overload. The addition of a dedicated auditory papilla in land vertebrates appears to have favored the separation of vestibular and auditory efferents and specializations for more sophisticated and more diverse functions.

Wnt signaling activates at least three different pathways involved in development and disease. Interactions of secreted ligands and inhibitors with cell-surface receptors result in the activation or regulation of particular downstream intracellular cascades. During the developmental stages of otic vesicle closure and beginning morphogenesis, the forming inner ear transcribes a plethora of Wnt-related genes. We report expression of 23 genes out of 25 tested in situ hybridization probes on tissue serial sections. Sensory primordia and Frizzled gene expression share domains, with Fzd1 being a continuous marker. Prospective nonsensory domains express Wnts, whose transcripts mainly flank prosensory regions. Finally, Wnt inhibitor domains are superimposed over both prosensory and nonsensory otic regions. Three Wnt antagonists, Dkk1, SFRP2, and Frzb are prominent. Their gene expression patterns partly overlap and change over time, which adds to the diversity of molecular micro-environments. Strikingly, prosensory domains express Wnts transiently. This includes (1) the prosensory otic region of high proliferation, neuroblast delamination, and programmed cell death at stage 20/21 (Wnt3,, and (2) sensory primordia at stage 25 (Wnt7a, Wnt9a). In summary, robust Wnt-related gene expression shows both spatial and temporal tuning during inner ear development as the otic vesicle initiates morphogenesis and prosensory cell fate determination.