Person:

Nishitani, Allison M

Lrig proteins are conserved transmembrane proteins that modulate a variety of signaling pathways from worm to humans. In mammals, there are three family members – Lrig1, Lrig2, and Lrig3 – that are defined by closely related extracellular domains with a similar arrangement of leucine rich repeats and immunoglobulin domains. However, the intracellular domains show little homology. Lrig1 inhibits EGF signaling through internalization and degradation of ErbB receptors. Although Lrig3 can also bind ErbB receptors in vitro, it is unclear whether Lrig2 and Lrig3 exhibit similar functions to Lrig1. To gain insights into Lrig gene functions in vivo, we compared the expression and function of the Lrigs in the inner ear, which offers a sensitive system for detecting effects on morphogenesis and function. We find that all three family members are expressed in the inner ear throughout development, with Lrig1 and Lrig3 restricted to subsets of cells and Lrig2 expressed more broadly. Lrig1 and Lrig3 overlap prominently in the developing vestibular apparatus and simultaneous removal of both genes disrupts inner ear morphogenesis. This suggests that these two family members act redundantly in the otic epithelium. In contrast, although Lrig1 and Lrig2 are frequently co-expressed, Lrig1−/−;Lrig2−/− double mutant ears show no enhanced structural abnormalities. At later stages, Lrig1 expression is sustained in non-sensory tissues, whereas Lrig2 levels are enhanced in neurons and sensory epithelia. Consistent with these distinct expression patterns, Lrig1 and Lrig2 mutant mice exhibit different forms of impaired auditory responsiveness. Notably, Lrig1−/−;Lrig2−/− double mutant mice display vestibular deficits and suffer from a more severe auditory defect that is accompanied by a cochlear innervation phenotype not present in single mutants. Thus, Lrig genes appear to act both redundantly and independently, with Lrig2 emerging as the most functionally distinct family member.

The vestibular system of the inner ear detects head position using three orthogonally oriented semicircular canals. Vestibular function relies on precise canal shape and orientation, and slight changes can cause vestibular defects. Canals are sculpted from pouches that protrude from the otic vesicle, the simple sphere of epithelium that forms the inner ear. In the center of each pouch, a "fusion plate" forms where cells lose their epithelial morphology and the basement membrane breaks down. The opposing layers of the fusion plate intercalate and are subsequently removed, creating a canal. Proper fusion depends on Netrin-1, which regulates basement membrane breakdown during fusion in mice, although the underlying molecular mechanism is unknown. This dissertation describes our work to better understand the cellular effects of Netrin-1 during canal formation. Although vestibular apparatus structure is shared among species, some developmental events that lead to this structure differ. For example, while fusion plate basement membrane breakdown is conserved, apoptosis is required for fusion in chicks, but not in mice. We used gain-of-function approaches to determine the main cellular effect of Netrin-1 during fusion in chicks and mice. We show that overexpression of Netrin-1 in chicks prevents canal fusion from occurring normally by interfering with apoptosis. On the other hand, we show that ectopic expression of Netrin-1 in mice using a conditional expression allele causes excessive fusion, resulting in canal truncation. This suggests that Netrin-1 may play divergent roles during canal morphogenesis in chicks and mice. To determine if Netrin-1 regulates the basement membrane in other contexts, we created a Netrin-1 conditional null allele. This was necessary because existing Netrin-1 mutants express residual Netrin-1 protein, which could be sufficient to rescue basement membrane defects in other tissues, and because existing mutants die shortly after birth, preventing postnatal analysis. Complete loss of Netrin-1 protein in our newly generated mice does not cause more severe defects in fusion compared to existing Netrin-1 hypomorphs, suggesting that residual Netrin-1 protein does not affect the basement membrane during fusion in Netrin-1 hypomorphs. Future work will determine if complete loss of Netrin-1 affects basement membrane integrity in other tissues.

Information flow through neural circuits is determined by the nature of the synapses linking the subtypes of neurons. How neurons acquire features distinct to each synapse remains unknown. We show that the transcription factor Mafb drives the formation of auditory ribbon synapses, which are specialized for rapid transmission from hair cells to spiral ganglion neurons (SGNs). Mafb acts in SGNs to drive differentiation of the large postsynaptic density (PSD) characteristic of the ribbon synapse. In Mafb mutant mice, SGNs fail to develop normal PSDs, leading to reduced synapse number and impaired auditory responses. Conversely, increased Mafb accelerates synaptogenesis. Moreover, Mafb is responsible for executing one branch of the SGN differentiation program orchestrated by the Gata3 transcriptional network. Remarkably, restoration of Mafb rescues the synapse defect in Gata3 mutants. Hence, Mafb is a powerful regulator of cell-type specific features of auditory synaptogenesis that offers a new entry point for treating hearing loss. DOI: http://dx.doi.org/10.7554/eLife.01341.001