Ultra-rapid drug delivery in the oral cavity using ultrasound.

The delivery of therapeutics to the gastrointestinal (GI) mucosa remains primarily a function of diffusion and rapid delivery is a significant goal in drug delivery science. However, delivery is hindered by the molecular barrier properties of the mucosa, as well as environmental factors. We hypothesized that low-frequency ultrasound can overcome these barriers, achieving rapid delivery in an engineered, clinically-relevant system for buccal administration. The hand-held system enabled delivery of macromolecules in short, 60-s treatment times ex vivo. Tolerability of the prototype was demonstrated in awake, (unsedated) dogs. Finally, this technology enhanced the efficacy of the anti-inflammatory agent, budesonide, allowing for prophylactic treatment in a hamster model of oral inflammatory lesions in vivo. The capacity to deliver therapeutics in a targeted and rapid manner in a clinically-relevant form-factor presents an intriguing capability to expand the repertoire of therapeutics that can be applied topically in the mouth and beyond.

Distinct functions for netrin 1 in chicken and murine semicircular canal morphogenesis.

The vestibular system of the inner ear detects head position using three orthogonally oriented semicircular canals; even slight changes in their shape and orientation can cause debilitating behavioral defects. During development, the canals are sculpted from pouches that protrude from the otic vesicle, the embryonic anlage of 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. Cells in the fusing epithelia intercalate and are removed, creating a canal. In mice, fusion depends on the secreted protein netrin 1 (Ntn1), which is necessary for basement membrane breakdown, although the underlying molecular mechanism is unknown. Using gain-of-function approaches, we found that overexpression of Ntn1 in the chick otic vesicle prevented canal fusion by inhibiting apoptosis. In contrast, ectopic expression of the same chicken Ntn1 in the mouse otic vesicle, where apoptosis is less prominent, resulted in canal truncation. These findings highlight the importance of apoptosis for tissue morphogenesis and suggest that Ntn1 may play divergent cellular roles despite its conserved expression during canal morphogenesis in chicken and mouse.

Fluorescent Protein Approaches in Alpha Herpesvirus Research.

In the nearly two decades since the popularization of green fluorescent protein (GFP), fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.

In vivo analysis of Lrig genes reveals redundant and independent functions in the inner ear.

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.

Cross-repressive interactions between Lrig3 and netrin 1 shape the architecture of the inner ear.

The sense of balance depends on the intricate architecture of the inner ear, which contains three semicircular canals used to detect motion of the head in space. Changes in the shape of even one canal cause drastic behavioral deficits, highlighting the need to understand the cellular and molecular events that ensure perfect formation of this precise structure. During development, the canals are sculpted from pouches that grow out of a simple ball of epithelium, the otic vesicle. A key event is the fusion of two opposing epithelial walls in the center of each pouch, thereby creating a hollow canal. During the course of a gene trap mutagenesis screen to find new genes required for canal morphogenesis, we discovered that the Ig superfamily protein Lrig3 is necessary for lateral canal development. We show that this phenotype is due to ectopic expression of the axon guidance molecule netrin 1 (Ntn1), which regulates basal lamina integrity in the fusion plate. Through a series of genetic experiments, we show that mutually antagonistic interactions between Lrig3 and Ntn1 create complementary expression domains that define the future shape of the lateral canal. Remarkably, removal of one copy of Ntn1 from Lrig3 mutants rescues both the circling behavior and the canal malformation. Thus, the Lrig3/Ntn1 feedback loop dictates when and where basement membrane breakdown occurs during canal development, revealing a new mechanism of complex tissue morphogenesis.

Early retinal activity and visual circuit development.

Does spontaneous retinal activity prior to vision play a role in the establishment of visual maps? In this issue of Neuron, two separate papers by Huberman et al. and Hooks and Chen demonstrate a role for early spontaneous retinal activity in the establishment of ocular dominance columns and synaptic refinement at retinogeniculate synapses.