Temporal and spatial coordination of multiple cell fate decisions is essential for proper organogenesis. Here, we define gene interactions that transform the neurogenic epithelium of the developing inner ear into specialized mechanosensory receptors. By Cre-loxP fate mapping, we show that vestibular sensory hair cells derive from a previously neurogenic region of the inner ear. The related bHLH genes Ngn1 (Neurog1) and Math1 (Atoh1) are required, respectively, for neural and sensory epithelial development in this system. Our analysis of mouse mutants indicates that a mutual antagonism between Ngn1 and Math1 regulates the transition from neurogenesis to sensory cell production during ear development. Furthermore, we provide evidence that the transition to sensory cell production involves distinct autoregulatory behaviors of Ngn1 (negative) and Math1 (positive). We propose that Ngn1, as well as promoting neurogenesis, maintains an uncommitted progenitor cell population through Notch-mediated lateral inhibition, and Math1 irreversibly commits these progenitors to a hair-cell fate.

Cochlear ganglion neurons communicate sound information from cochlear hair cells to auditory brainstem neurons through precisely wired circuits. Understanding auditory circuit assembly is a significant challenge because of the small size of the otic vesicle and difficulties labeling and imaging embryonic neurons. We used genetic fate mapping in the mouse to visualize the morphologies of individual cochlear ganglion neurons throughout development, from their origin in the Neurogenin1-positive neurogenic domain in the otic vesicle to the formation of connections with targets in the cochlea and in the cochlear nucleus. We found that auditory neurons with different patterns of connectivity arise from discrete populations of Neurogenin1-positive precursors that make stereotyped wiring decisions depending on when and where they are born. Auditory precursors are segregated from vestibular precursors early in neurogenesis. Within this population, cochlear ganglion neurons with type I and type II morphologies are apparent before birth and develop within common pools of precursors. The peripheral projections are initially complex and branched and then become simple and straight after reaching the edge of the sensory epithelium. Subsequently, a small number of projections attain obvious type II morphologies, beginning at embryonic day 16.5 (E16.5), when hair cells begin to differentiate. Centrally, cochlear ganglion axons are topographically organized in the auditory brainstem as early as E15.5, when the cochlear nucleus is still immature. These findings suggest that Neurogenin1 precursors possess intrinsic programs of differentiation that direct early auditory circuit assembly events before the maturation of presynaptic and postsynaptic target cells.

The evolution of the ancestral eukaryotic flagellum is an example of a cellular organelle that became dispensable in some modern eukaryotes while remaining an essential motile and sensory apparatus in others. To help define the repertoire of specialized proteins needed for the formation and function of cilia, we used comparative genomics to analyze the genomes of organisms with prototypical cilia, modified cilia, or no cilia and identified approximately 200 genes that are absent in the genomes of nonciliated eukaryotes but are conserved in ciliated organisms. Importantly, over 80% of the known ancestral proteins involved in cilia function are included in this small collection. Using Drosophila as a model system, we then characterized a novel family of proteins (OSEGs: outer segment) essential for ciliogenesis. We show that osegs encode components of a specialized transport pathway unique to the cilia compartment and are related to prototypical intracellular transport proteins.

The majority of genes of multicellular organisms encode proteins with functions that are not required for viability but contribute to important physiological functions such as behavior and reproduction. It is estimated that 75% of the genes of Drosophila melanogaster are nonessential. Here we report on a strategy used to establish a large collection of stocks that is suitable for the recovery of mutations in such genes. From ‫000,27ف‬ F 3 cultures segregating for autosomes heavily treated with ethyl methanesulfonate (EMS), ‫000,21ف‬ lines in which the treated second or third chromosome survived in homozygous condition were selected. The dose of EMS induced an estimated rate of 1.2-1.5 ϫ 10 Ϫ3 mutations/gene and predicts five to six nonessential gene mutations per chromosome and seven to nine alleles per locus in the samples of 6000 second chromosomes and 6000 third chromosomes. Due to mosaic mutations induced in the initial exposure to the mutagen, many of the lines are segregating or are now fixed for lethal mutations on the mutagenized chromosome. The features of this collection, known as the Zuker collection, make it a valuable resource for forward and reverse genetic screens for mutations affecting a wide array of biological functions.T HE search for mutations in genes whose protein generated and now maintain a collection of 12,336 lines with EMS-treated chromosomes, with 6079 lines with products are known is complicated by the uncertainty regarding the phenotypes expected for such mumutagenized second chromosomes and 6257 lines with mutagenized third chromosomes. Here we describe the tations. To search for mutations in a gene encoding a protein of interest, an F 2 screen can be used if a mutant strategy used to generate this collection and report on the features that make it a valuable resource for genetic or chromosome deficiency is available for the locus. In analyses of a wide variety of physiological processes in the absence of a chromosome deficiency, it is necessary Drosophila. to carry out an F 3 screen to produce flies homozygous for a mutagenized chromosome. The interest in this laboratory has been in genes encoding eye-specific pro-MATERIALS AND METHODS teins, the mutations of which are expected to result in viable adults. In the past this approach has been effective Deciding on the optimal level of EMS required us to strive in recovering mutations of a number of genes; however, for a dose high enough to engender a high mutation rate, each recovery required a large F 3 screen involving thoubut not so high as to produce too many homozygous lethal chromosomes. We arbitrarily settled on the criterion that at sands of individual lines (Table 1). least 75% of the treated chromosomes in a run must be homoOn the basis of our knowledge of the Drosophila zygous lethal. The reliable achievement of 75% lethality rephototransduction cascade, we knew that mutations for quired experimentation with mutagenic protocols, in the proevery gene had not yet been generated and that a definicess of which many F ...