Generating mouse models of degenerative diseases using Cre/lox-mediated in vivo mosaic cell ablation.

Most degenerative diseases begin with a gradual loss of specific cell types before reaching a threshold for symptomatic onset. However, the endogenous regenerative capacities of different tissues are difficult to study, because of the limitations of models for early stages of cell loss. Therefore, we generated a transgenic mouse line (Mos-iCsp3) in which a lox-mismatched Cre/lox cassette can be activated to produce a drug-regulated dimerizable caspase-3. Tissue-restricted Cre expression yielded stochastic Casp3 expression, randomly ablating a subset of specific cell types in a defined domain. The limited and mosaic cell loss led to distinct responses in 3 different tissues targeted using respective Cre mice: reversible, impaired glucose tolerance with normoglycemia in pancreatic ß cells; wound healing and irreversible hair loss in the skin; and permanent moderate deafness due to the loss of auditory hair cells in the inner ear. These mice will be important for assessing the repair capacities of tissues and the potential effectiveness of new regenerative therapies.

Lhx3, a LIM domain transcription factor, is regulated by Pou4f3 in the auditory but not in the vestibular system.

A dominant mutation of the gene encoding the POU4F3 transcription factor underlies human non-syndromic progressive hearing loss DFNA15. Using oligonucleotide microarrays to generate expression profiles of inner ears of Pou4f3(ddl/ddl) mutant and wild-type mice, we have identified and validated Lhx3, a LIM domain transcription factor, as an in vivo target gene regulated by Pou4f3. Lhx3 is a hair cell-specific gene expressed in all hair cells of the auditory and vestibular system as early as embryonic day 16. The level of Lhx3 mRNA is greatly reduced in the inner ears of embryonic Pou4f3 mutant mice. Our data also show that the expression of Lhx3 is regulated differently in auditory and vestibular hair cells. This is the first example of a hair cell-specific gene expressed both in auditory and in vestibular hair cells, with differential regulation of expression in these two closely related systems.

Brn3c null mutant mice show long-term, incomplete retention of some afferent inner ear innervation.

BACKGROUND: Ears of Brn3c null mutants develop immature hair cells, identifiable only by certain molecular markers, and undergo apoptosis in neonates. This partial development of hair cells could lead to enough neurotrophin expression to sustain sensory neurons through embryonic development. We have therefore investigated in these mutants the patterns of innervation and of expression of known neurotrophins. RESULTS: At birth there is a limited expression of BDNF and NT-3 in the mutant sensory epithelia and DiI tracing shows no specific reduction of afferents or efferents that resembles neurotrophin null mutations. At postnatal day 7/8 (P7/8), innervation is severely reduced both qualitatively and quantitatively. 1% of myosin VIIa-positive immature hair cells are present in the mutant cochlea, concentrated in the base. Around 20% of immature hair cells exist in the mutant vestibular sensory epithelia. Despite more severe loss of hair cells (1% compared to 20%), the cochlea retains many more sensory neurons (46% compared to 15%) than vestibular epithelia. Even 6 months old mutant mice have some fibers to all vestibular sensory epithelia and many more to the cochlear apex which lacks MyoVIIa positive hair cells. Topologically organized central cochlea projections exist at least until P8, suggesting that functional hair cells are not required to establish such projections. CONCLUSION: The limited expression of neurotrophins in the cochlea of Brn3c null mice suffices to support many sensory neurons, particularly in the cochlea, until birth. The molecular nature of the long term survival of apical spiral neurons remains unclear.