Hes1 and Hes5 activities are required for the normal development of the hair cells in the mammalian inner ear.

The mammalian inner ear contains two sensory organs, the cochlea and vestibule. Their sensory neuroepithelia are characterized by a mosaic of hair cells and supporting cells. Cochlear hair cells differentiate in four rows: a single row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs). Recent studies have shown that Math1, a mammalian homolog of Drosophila atonal is a positive regulator of hair cell differentiation. The basic helix-loop-helix (bHLH) genes Hes1 and Hes5 (mammalian hairy and Enhancer-of-split homologs) can influence cell fate determination by acting as negative regulators to inhibit the action of bHLH-positive regulators. We show by using reverse transcription-PCR analysis that Hes1, Hes5, and Math1 are expressed in the developing mouse cochleae. In situ hybridization revealed a widespread expression of Hes1 in the greater epithelial ridge (GER) and in lesser epithelial ridge (LER) regions. Hes5 is predominantly expressed in the LER, in supporting cells, and in a narrow band of cells within the GER. Examination of cochleae from Hes1(-/-) mice showed a significant increase in the number of IHCs, whereas cochleae from Hes5(-/-) mice showed a significant increase in the number of OHCs. In the vestibular system, targeted deletion of Hes1 and to a lesser extent Hes5 lead to formation of supernumerary hair cells in the saccule and utricle. The supernumerary hair cells in the mutant mice showed an upregulation of Math1. These data indicate that Hes1 and Hes5 participate together for the control of inner ear hair cell production, likely through the negative regulation of Math1.

Embryonic development of the Drosophila brain: formation of commissural and descending pathways.

The establishment of initial axonal pathways in the embryonic brain of Drosophila melanogaster was investigated at the cellular and molecular level using antibody probes, enhancer detector strains and axonal pathfinding mutants. During embryogenesis, two bilaterally symmetrical cephalic neurogenic regions form, which are initially separated from each other and from the ventral nerve cord. The brain commissure that interconnects the two brain hemispheres is pioneered by axons that project towards the midline in close association with an interhemispheric cellular bridge. The descending longitudinal pathways that interconnect the brain to the ventral nerve cord are prefigured by a chain of longitudinal glial cells and a cellular bridge between brain and subesophageal ganglion; pioneering descending and ascending neurons grow in close association with these structures. The formation of the embryonic commissural and longitudinal pathways is dependent on cells of the CNS midline. Mutations in the commissureless gene, which affects growth cone guidance towards the midline, result in a marked reduction of the brain commissure. Mutations in the single-minded gene and in other spitz group genes, which affect the differentiation of CNS midline cells, result in the absence or aberrant projection of longitudinal pathways. The analysis of axon pathway formation presented here reveals remarkable similarities as well as distinct differences in the embryonic development of the brain and the segmental ganglia, and forms the basis for a comprehensive genetic and molecular genetic dissection of axonal pathfinding processes in the developing brain.

Developmental defects in brain segmentation caused by mutations of the homeobox genes orthodenticle and empty spiracles in Drosophila.

We have studied the roles of the homeobox genes orthodenticle (otd) and empty spiracles (ems) in embryonic brain development of Drosophila. The embryonic brain is composed of three segmental neuromeres. The otd gene is expressed predominantly in the anterior neuromere; expression of ems is restricted to the two posterior neuromeres. Mutation of otd eliminates the first (protocerebral) brain neuromere. Mutation of ems eliminates the second (deutocerebral) and third (tritocerebral) neuromeres. otd is also necessary for development of the dorsal protocerebrum of the adult brain. We conclude that these homeobox genes are required for the development of specific brain segments in Drosophila, and that the regionalized expression of their homologs in vertebrate brains suggests an evolutionarily conserved program for brain development.

Axogenesis in the embryonic brain of the grasshopper Schistocerca gregaria: an identified cell analysis of early brain development.

Axogenesis in the embryonic brain was studied at the single cell level in the grasshopper Schistocerca gregaria. A small set of individually identifiable pioneer neurons establishes a primary axon scaffold during early embryogenesis. At the beginning of scaffold formation, pioneering axons navigate along and between glial borders that surround clusters of proliferating neuroblasts. In each brain hemisphere, an axonal outgrowth cascade involving a series of pioneer neurons establishes a pathway from the optic ganglia to the brain midline. At the midline the primary preoral commissural interconnection in the embryonic brain is pioneered by a pair of midline-derived pioneer neurons. A second preoral commissural connection is pioneered by two pairs of pars intercerebralis pioneer neurons. Descending tracts are pioneered by the progeny of identified neuroblasts in the pars intercerebralis, deutocerebrum and tritocerebrum; the postoral tritocerebral commissure is pioneered by a pair of tritocerebral neurons. All of the pioneering brain neurons express the cell adhesion molecule fasciclin I during initial axon outgrowth and fasciculation. Once established, the primary axon scaffold of the brain is used for fasciculation by subsequently differentiating neurons and, by the 40% stage of embryogenesis, axonal projections that characterize the mature brain become evident. The single cell analysis of grasshopper brain development presented here sets the stage for manipulative cell biological experiments and provides the basis for comparative molecular genetic studies of embryonic brain development in Drosophila.

The gene poxn controls different steps of the formation of chemosensory organs in Drosophila.

The gene poxn codes for a transcriptional regulator that specifies poly-innervated (chemosensory), as opposed to mono-innervated (mechanosensory), organs in Drosophila. The ectopic expression of poxn during metamorphosis results in a transformation of the morphology and central projection of adult mechanosensory organs toward those of chemosensory organs. Here we show, by electron microscopy analysis of normal and transformed bristles and by Dil labeling of the innervating neurons, that poxn also controls the number of neurons. To determine whether poxn can transform not only the sense organ precursor cells but also their daughter cells, we examine the effects of the ectopic expression of poxn at different stages of the lineage, and we conclude that poxn can act at a late stage to affect the fate of the undifferentiated neuron.

Developmental expression of TERM-1 glycoprotein on growth cones and terminal arbors of individual identified neurons in the grasshopper.

The chemoaffinity theory postulates the existence of cell-specific molecular signals that uniquely identify individual developing neurons. Such molecules are thought to promote both accurate axon outgrowth and the formation of correct synaptic connections. To identify candidates for such neuron-specific recognition molecules, we generated monoclonal antibodies that recognize surface-associated antigens expressed by individual identified neurons in the grasshopper embryo. Here we report on a molecular label that is expressed exclusively by two pairs of sibling interneurons in the developing CNS. Our experiments indicate that during axogenesis, this molecule is expressed at the surface of the growth cones of these cells, while during subsequent synaptogenesis, it becomes concentrated at the cells' developing terminal arbors. In both cases the molecule appears to be secreted by the labeled structures. This molecule, which we call TERM-1, is a glycoprotein with a molecular weight of approximately 48 kDa. The highly restricted spatiotemporal expression pattern of TERM-1 implies that individual developing neurons can acquire and retain unique molecular labels that may be important for neuron-specific outgrowth and target recognition.