Hox genes and tonotopic organization of auditory brainstem circuits

The formations of functional neuronal circuits are achieved through multiple developmental processes, beginning at neuronal progenitor specification and establishment of topographic connectivity to refinement of topographic circuits and synaptic maturation of the circuits. Though, most of the underlying neuronal connectivity in different circuits have been identified, the molecular mechanisms guiding the establishment and refinement of their input-output topographic relay, are largely unknown. Refinement and maturation of topographic connectivity is essential in the visual system (Huberman et al., 2008), the somatosensory system (Fox et al., 2005) as well as in the auditory system (Kandler et al., 2009). During my Ph.D, I studied two different neuronal circuits, one addressing the development of tonotopic organization in auditory sensory circuits; and the other unraveling the neuroanatomical pathways in whisker related sensori-motor interactions and whisker movements.
The auditory system has a unique topographic organization, such that all auditory nuclei represent a gradient of frequencies and two neighboring bands of neurons respond to neighboring sound frequencies. Such an organization with an orderly representation of frequencies is called tonotopy and tonotopic organization is essential for efficient discrimination of sound frequencies (Kandler et al., 2009). The tonotopic organization of the auditory nuclei are considered to be developmentally hardwired, however, elaborate processes of refinement are essential to achieve the precision of the adult tonotopic circuits (Kandler et al., 2009; Clause et al., 2014). The brainstem auditory circuits, which consist of the cochlear nucleus (CN) and the superior olivary complex (SOC) are also tonotopically organized. The CN is further subdivided into the anterior ventral cochlear nucleus (AVCN), posterior ventral cochlear nucleus (PVCN) and the dorsal cochlear nucleus (DCN). The AVCN arises from rhombomeric progenitor zones, r2-r3, which are characterized by the combinatorial expression of Hox paralogous group 2 genes (Hox PG2), Hoxa2 and Hoxb2 (Narita and Rijli, 2009; Di Bonito et al., 2013). Hox genes are determinants of topographic information and influence topographic organization as well as topographic input-output connectivity of several hindbrain nuclei (Philippidou and Dasen, 2013). In our present study, we investigate the role of Hox PG2 genes in the tonotopic organization of the brainstem auditory circuits, with focus on AVCN.
Our results suggest an essential role of Hox PG2 genes in the maturation and refinement of the tonotopic organization and connectivity of the AVCN. Using conditional deletions of Hox PG2 genes targeting the post-mitotic bushy cells in the AVCN, we show that the gross tonotopic organization of the AVCN, which is established very early during development, is unaffected. However, processes involving refinement of the tonotopic organization are impaired in the absence of the Hox PG2 genes. In the Hox PG2 mutants, peripheral afferents of the spiral ganglion (SG) neurons target less precisely, resulting in a broader spread of targeting bands in the AVCN. These aberrant SG projections are not developmentally refined and are still maintained in the adult mutants as observed in the auditory pure tone stimulation experiments. Pure tone auditory stimulations activate broader bands with larger number of activated neurons in the mutants. The broadening of the activated bands leads to reduced separation (also overlap) between bands of activated neurons responding to two neighboring sound frequencies. This results in a decreased resolution of the tonotopic organization in the AVCN, affecting sound frequency discrimination in the Hox PG2 conditional mutants. In an auditory tone based discriminating fear conditioning experiment, the Hox PG2 mutants are unable to distinguish between two close sound frequencies, compared to the controls. To explore the molecular mechanisms underlying the described phenotype, we performed a transcriptome analysis on the mutant AVCN bushy cells. Our results showed a deregulation of acvitity associated genes and synapse associated genes in the absence of Hox PG2 genes. Thus, we looked into the development of synapses between the SG afferents and the AVCN bushy cells, the giant Endbulb of Held synapses. The Endbulb of Held synapse maturation occurs in an activity dependent manner involving elimination of multi-axonal inputs to retain 1-2 major inputs, in the weeks after hearing onset. Our analysis showed that synaptic maturation of the Endbulb synapses were affected and the mutant Endbulbs receive higher numbers of SG axonal inputs. Thus, our results show that conditional deletion of Hox PG2 genes in specific subsets of AVCN neurons affects several late developmental refinement processes, culminating in loss of resolution of tonotopic precision and reduction in sound frequency discrimination.
In addition to the above described study, this thesis manuscript also includes another study (currently in press, European Journal of Neuroscience) done in collaboration with Varun Sreenivasan from Prof. Carl C. Petersen’s group, EPFL, Switzerland. In this study, we map the neuronal pathways connecting cortical inputs to hindbrain facial motor nucleus (FMN), driving peripheral facial muscles in the mouse whisker system. We investigate how cortical inputs from motor cortex (M1) and somatosensory cortex (S1) interact with premotor and motor nuclei in the hindbrain, while driving different whisker movements. We identify distinct subsets of premotor nuclei associated with whisker retraction and whisker protraction, which receive differential cortical inputs from S1 and M1, respectively. Our results suggest two parallel pathways through which M1 driven whisker protraction and S1 driven whisker retraction are actuated. Thus, in this study, we further the understanding of the anatomical pathways underlying whisker movements.