A noncoding point mutation of Zeb1 causes multiple developmental malformations and obesity in Twirler mice

Heterozygous Twirler (Tw) mice develop obesity and circling behavior associated with malformations of the inner ear, whereas homozygous Tw mice have cleft palate and die shortly after birth. Zeb1 is a zinc finger protein that contributes to mesenchymal cell fate by repression of genes whose expression defines epithelial cell identity. This developmental pathway is disrupted in inner ears of Tw/Tw mice. The purpose of our study was to comprehensively characterize the Twirler phenotype and to identify the causative mutation. The Tw/+ inner ear phenotype includes irregularities of the semicircular canals, abnormal utricular otoconia, a shortened cochlear duct, and hearing loss, whereas Tw/Tw ears are severely malformed with barely recognizable anatomy. Tw/+ mice have obesity associated with insulin-resistance and have lymphoid organ hypoplasia. We identified a noncoding nucleotide substitution, c.58+181G>A, in the first intron of the Tw allele of Zeb1 (Zeb1(Tw)). A knockin mouse model of c.58+181G>A recapitulated the Tw phenotype, whereas a wild-type knockin control did not, confirming the mutation as pathogenic. c.58+181G>A does not affect splicing but disrupts a predicted site for Myb protein binding, which we confirmed in vitro. In comparison, homozygosity for a targeted deletion of exon 1 of mouse Zeb1, Zeb1(ΔEx1), is associated with a subtle abnormality of the lateral semicircular canal that is different than those in Tw mice. Expression analyses of E13.5 Twirler and Zeb1(ΔEx1) ears confirm that Zeb1(ΔEx1) is a null allele, whereas Zeb1(Tw) RNA is expressed at increased levels in comparison to wild-type Zeb1. We conclude that a noncoding point mutation of Zeb1 acts via a gain-of-function to disrupt regulation of Zeb1(Tw) expression, epithelial-mesenchymal cell fate or interactions, and structural development of the inner ear in Twirler mice. This is a novel mechanism underlying disorders of hearing or balance.     

A Myo6 mutation destroys coordination between the myosin heads, revealing new functions of myosin VI in the stereocilia of mammalian inner ear hair cells

Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or ‘gating’ in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI–impaired hair cells, and ultimately leading to deafness.     

Predicting class II MHC/peptide multi-level binding with an iterative stepwise discriminant analysis meta-algorithm.

MOTIVATION: Predicting peptides that bind to both Major Histocompatibility Complex (MHC) molecules and T cell receptors provides crucial information for vaccine development. An agretope is that portion of a peptide that interacts with an MHC molecule. The identification and prediction of agretopes is the first step towards vaccine design. RESULTS: An iterative stepwise discriminant analysis meta-algorithm is utilized to derive a quantitative motif for classifying potential agretopes as high-, moderate- or non-binders for HLA-DR1, a class II MHC molecule. A large molecular online database provides the input for this data-driven algorithm. The model correctly classifies over 85% of the peptides in the database. AVAILABILITY: Stepwise discriminant analysis software is available commercially in SPSS and BMDP statistical software packages. Peptides known to bind MHC molecules can be downloaded from http://wehih.wehi.edu.au/mhcpep/. Peptides known not to bind HLA-DR1 are available from the author upon request. CONTACT: ronna@ucsfresno.edu.     

Lineage-tracing and translatomic analysis of damage-inducible mitotic cochlear progenitors identifies candidate genes regulating regeneration

Cochlear supporting cells (SCs) are glia-like cells critical for hearing function. In the neonatal cochlea, the greater epithelial ridge (GER) is a mitotically quiescent and transient organ, which has been shown to nonmitotically regenerate SCs. Here, we ablated Lgr5⁺ SCs using Lgr5-DTR mice and found mitotic regeneration of SCs by GER cells in vivo. With lineage tracing, we show that the GER houses progenitor cells that robustly divide and migrate into the organ of Corti to replenish ablated SCs. Regenerated SCs display coordinated calcium transients, markers of the SC subtype inner phalangeal cells, and survive in the mature cochlea. Via RiboTag, RNA-sequencing, and gene clustering algorithms, we reveal 11 distinct gene clusters comprising markers of the quiescent and damaged GER, and damage-responsive genes driving cell migration and mitotic regeneration. Together, our study characterizes GER cells as mitotic progenitors with regenerative potential and unveils their quiescent and damaged translatomes.

Cochlear supporting cells are glia-like cells essential for hearing. Genetic ablation of Lgr5+ supporting cells reveals a population of damage inducible, mitotically activated progenitors in the greater epithelial ridge, which can divide and migrate into the organ of Corti to replenish ablated supporting cells. Translatomic analyses provide insights into the genes that may regulate this regenerative potential.     

Iterative stepwise discriminant analysis: a meta-algorithm for detecting quantitative sequence motifs.

An algorithm is presented for detecting a quantitative pattern in peptide fragments that bind class II major histocompatibility complex (MHC) molecules. It is referred to as a meta-algorithm because it requires successive applications of Stepwise Discriminate Analysis (SDA). On every iteration the best subsequence candidates are selected from sequences known to bind class II MHC molecules. When SDA compares probable binding subsequences with subsequences known not to bind class II MHC molecules, a quantitative model emerges that is capable of classifying subsequences as binding or non-binding. In an iterative manner, the resultant model is utilized as a criterion for selecting probable binding subsequence candidates. The procedure is repeated until models converge. In the illustrated examples, the final models correctly classify over 95% of the peptides in a database of peptides whose binding affinity for HLA-DR1 is known. The final model can then be used to predict the binding affinity of peptides that have not yet been laboratory tested.