Jason Tait Sanchez, Assistant Professor at Northwestern University provides insight into why the genetically modified chicken is a sound approach to the study of hearing. We discover how tonotopic properties are established in the chicken auditory brainstem by using novel and innovated genetic research methods
It is well established that the chicken is a valuable research tool to study basic biological questions in numerous health-related areas, including immunology and infectious diseases(1,2).
Recent applications of gene editing in chickens also suggests an innovative era is on the horizon for developmental and sensory neurobiology as well(3,4). With respect to hearing development, mammals and birds share comparable auditory functions at the cellular, synaptic, and neural circuit level(5,6) and both species encode sound similarly across the frequency axis, a process known as tonotopic tuning(7,8).
Tonotopy is the spatial arrangement of where sounds of different frequencies are processed. Tonotopy originates along the peripheral sensory epithelium and is preserved throughout the entire auditory system. Tonotopy in the central auditory pathway is arranged not only by the specific locations of neurons and their inputs, but by differences in their structural and functional properties along the tonotopic axis(9,10). Exemplars of this are found in the mammalian anteroventral cochlear nucleus and the chicken cochlear nucleus magnocellularis (NM), which are analogous, first-order auditory brainstem structures. In this article, I will provide recent insight into how tonotopic properties are established in the chicken auditory brainstem by using novel and innovated genetic research methods.
Development of tonotopic properties
Despite more than a half-century of work on the development of tonotopic properties in the peripheral auditory system (i.e., the cochlea), little is known about its establishment in the central auditory system(11). This fundamental lack of knowledge is noteworthy, and several questions warrant discussion.
First, do tonotopic properties emerge from indiscriminate connections, or are there precise projections early in development? If precision exists early on, does refinement improve with maturation? Anatomical evidence argues that the topography of connections between the periphery and central pathway develops with considerable precision, well before hearing onset, and with substantial refinement thereafter(12-18).
Second, what role, if any, does spontaneous neuronal activity – as opposed to sound-driven activity – have on the development or maintenance of tonotopic properties? Physiological studies show that early in development, functional mapping along the tonotopic axis supports precise tuning independent of sound-driven activity (19-23).
Finally, what are the molecular and cellular signals responsible for establishing and maintaining distinct neuronal phenotypes along the tonotopic axis in the central pathway? The answer to this final question remains elusive, making it a significant and timely problem in developmental and sensory neurobiology in general(3).
Potential genes-of-interest
One thing is clear, however; both presynaptic axons and postsynaptic target neurons express genes – like neurotrophins – that may be responsible for establishing tonotopic properties in the central pathway. Neurotrophins, along with their cognate receptors, are growth factor proteins that support numerous aspects of normal nervous system development(24-27), and irregular neurotrophin signalling is implicated in pathophysiological conditions in both the peripheral and central nervous systems(28-30). This makes them a critical factor that promotes normal and abnormal biologically relevant properties(31).
The idea that neurotrophin signalling is important for the tonotopic establishment in the auditory system is supported by the following observations from the chicken NM.
(A) Schematic of electroporation in the hindbrain of a Hamburger Hamilton (HH) stage 12 chicken embryo. The injection pipette is filled with plasmid DNA and fast green dye for visualisation of the injected hindbrain (rhombomeres 5/6 [R5/R6]). In this study, a plasmid that coexpressed the gene-of-interest and the yellow fluorescent protein (YFP) reporter was used. Electroporated embryos are further incubated until the desired developmental stage is reached. (B) Embryonic (E) day 4 chicken showing the site of YFP expression relative to the left eye (arrow, E) and left otocyst (arrow, O). The dashed white line represents the dorsal brain and brainstem border. Scale bar = 480 μm. (C & C1). Brainstem slice (300 μm thick) from an E12 chicken under differential interference contrast (DIC, C) and fluorescent (YFP, C1) illumination. Dashed white lines represent the borders of nucleus magnocellularis (NM) and nucleus laminaris (NL). Note, the brainstem slice shows only one side of the tissue. White arrowheads represent midline cleft of the brainstem slice. White arrows show YFP expressing regions of NM and NL. V = ventral, M = medial. Scale bar = 240 μm. (D & D1). High magnification (80X water immersion objective) of an E18 chicken brainstem slice containing NM. The classic adendritic cell bodies37 are clearly visible under DIC illumination (upward arrow, D). With fluorescent illumination for the same slice, a transfected NM neuron identified by YFP fluorescence is clearly visible (upward arrow, D1). Asterisks = YFP expressing NM neurons just below the focal plane. Scale bar = 30 μm. Figure from Lu et al., 201748.
Second, the expression pattern of a very specific neurotrophin receptor (known as TrkB) is spatially and temporally dynamic; TrkB is present at embryonic (E) day 7, significantly reduced by E15 and absent at hatch (E21), but only in mid- to high-frequency regions(33).
Third, this expression pattern parallels a developmental period when functional properties are also differentially established along the tonotopic axis(34-37) and coincides with hearing maturation(38).
Finally, genetically modified maintenance of TrkB receptors in mid- to high-frequency NM prevents dendrite retraction and promotes aberrant neuronal excitability(39), properties that more closely resemble their low-frequency neuronal counterparts(10,40). The dynamic expression pattern of TrkB receptors regulates the development of distinct tonotopic properties found in NM and strongly supports the hypothesis that neurotrophin signalling establishes different neuronal phenotypes along the tonotopic axis in the central auditory pathway.
Why the chicken?
The chicken is an ideal model system over other mammalian research tools because they have tonotopic properties more commonly shared with humans. Chickens, like humans, utilise both low- and high-frequency sounds to perform behaviourally relevant auditory tasks, such as sound localisation and signal discrimination(41,42). This is unlike most mammalian research models, such as mice and rats, which rely primarily on ultrasonic hearing.
With respect to the development of hearing acuity, chickens (like humans) are also precocious animals. The chicken’s auditory system is near functional maturity at hatch, and the onset and refinement of hearing occur during embryonic stages38. This is unlike other low-frequency hearing mammalian research models (e.g., gerbil, guinea pig), whose hearing emerges ~10-16 days postnatal(43) and are susceptible to extrinsic factors that influence development.
Finally, the spatial and temporal expression of limited neurotrophin factors in the chicken NM(33,44) provides a novel opportunity to evaluate highly-specific neurotrophin signalling and its role in establishing neuronal topology. This is unlike the mammalian auditory system, which expresses many more neurotrophin factors across numerous developmental periods(45), ultimately confounding the study of neurotrophin signalling in regulating tonotopic development in these species.
A sound approach
Electroporation is a method that introduces genes into biologically relevant organisms like the chicken embryo. In ovo electroporation is a formidable tool to study neuron-specific development in the auditory brainstem(3,46). It permits the over-expression or knock-down of specific genes-of-interest (like neurotrophin factors) in order to analyse in vivo gene function(39,47). We and others have recently introduced genetic methods to obtain focal, stable and temporally regulated transgene expression of neurotrophins at multiple stages of chicken embryo development(3,39,48) (Fig. 1). It is advantageous over mammalian model systems for several reasons. First and foremost, because electroporation is performed in ovo, it permits gene expression in a normally developing biological system.
Second, genes are focally injected, allowing spatial control of expression in highly specific brain regions(49). Third, genes are temporally regulated by drug applications, enabling expression at precise developmental time periods(39,48).
Finally, genes are only expressed by a subset of neurons, allowing non-transfected neurons to serve as internal controls. This provides a rigorous and quantitative comparison of the neuron-autonomous effects of gene expression. The in ovo electroporation technique – together with either biochemical, pharmacological, and or in vivo functional assays – provides a genetic approach to study auditory neuron development associated with tonotopic differences in neuronal structure and function, as well as associated pathophysiological phenomena.
Indeed, a better understanding of normal auditory circuit assembly – along with unique structural and functional properties associated with tonotopic gradients – will provide a significant foundation for developing stem cell-based therapies for auditory-related disorders. However, stem cell-derived auditory neurons will only prove useful – therapeutically – if they are able to re-create neuronal properties that are characteristic of normal circuit maturation(50). A careful characterisation of neurotrophin signalling, the underlying molecular mechanism by which it operates, the role it plays in establishing normal neuronal properties, and the functional consequence of altering this biological process is necessary and appropriate.
Our research aims at addressing these issues by providing a comprehensive understanding of neurotrophin signalling and its role in establishing neuronal phenotypes along the tonotopic axis in the developing auditory brainstem, a biologically relevant structure which is essential for sound processing.
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Jason Tait Sanchez
Assistant Professor
Northwestern University
Tel: +1 847 491 4648
