Donna M. Fekete

Manipulating gene expression with replication-competent retroviruses.

Publisher Summary The obvious advantages of the chick for embryological studies have been tempered by the difficulties of genetic analyses in this organism. The ability to manipulate gene expression in the mouse embryo has rendered it the organism of choice for genetic studies, but the difficulty of physically manipulating the mammalian embryo limits the combination of these approaches to study development. The use of retroviral vectors to alter gene expression has proven to be a valuable alternative to transgenic approaches. Many of the genetic manipulations that have proven so powerful in the mouse can be mimicked with retroviral vectors. The use of retroviruses to alter gene expression has several advantages stemming from the comparative ease with which different expression patterns may be generated with the same virus. Many genes are reused in different tissues and at different times to mediate development. By varying the infection protocol, a single high titer virus stock may be used to address the role of a gene in many different developmental decisions.

Revisiting cell fate specification in the inner ear.

Generating the diversity of cell types in the inner ear may require an interplay between regional compartmentalization and local cellular interactions. Recent evidence has come from gene targeting, lineage analysis, fate mapping and gene expression studies. Notch signaling and neurogenic gene regulation are involved in patterning or specification of sensory organs, ganglion cells and hair cell mechanoreceptors.

Replication-competent retroviral vectors encoding alkaline phosphatase reveal spatial restriction of viral gene expression/transduction in the chick embryo

Replication-competent avian retroviruses, capable of transducing and expressing up to 2 kb of nonviral sequences, are now available to effect widespread gene transfer in chicken (chick) embryos (S. H. Hughes, J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave, J. Virol. 61:3004-3012, 1987). We have constructed novel avian retroviral vectors that encode human placental alkaline phosphatase as a marker whose expression can be histochemically monitored. These vectors have been tested for expression by introducing them into the embryonic chick nervous system. They have revealed that the expression of retrovirally transduced genes can be spatially and temporally limited without the need for tissue-specific promoters. By varying the site and time of infection, targeted gene transfer can be confined to selected populations of neural cells over the course of several days, a time window that is sufficient for many key developmental processes. The capability of differentially infecting specific target populations may avoid confounding variables such as detrimental effects of a transduced gene on processes unrelated to the cells or tissue of interest. These vectors and methods thus should be useful in studies of the effect of transduced genes on the development of various organs and tissues during avian embryogenesis. In addition, the vectors will facilitate studies aimed at an understanding of viral infection and expression patterns.

Cell fate specification in the inner ear.

From its origin as a single ectodermal patch, the inner ear becomes a labyrinth of chambers housing six to eight sensory organs. Along the way, specific cell fates are realized. The secrets underlying these cell fate specifications are beginning to be revealed through the application of several molecular-genetic approaches. Recent papers describing such approaches have included gene expression studies in the early otic epithelium and inner ear sensory epithelia. large-scale screens of zebrafish mutants to identify ear defects, and targeted gene perturbations of neurotrophins. of their receptors or of the Brn-3.1 transcription factor in mice.

Development of the vertebrate ear: insights from knockouts and mutants

The three divisions of the ear (outer, middle and inner) each have an important role in hearing, while the inner ear is also crucial for the sense of balance. How these three major components arise and coalesce to form the peripheral elements of the senses of hearing and balance is now being studied using molecular-genetic approaches. This article summarizes data from studies of knockout and mutant animals in which one or more divisions of the ear are abnormal. The data confirm that development of all three divisions of the ear depends on the genes involved in hindbrain segmentation and segment identity. Genes that are regionally expressed in the inner ear can, when absent or mutated, yield selective ablation of specific inner-ear structures or cell types.

MicroRNAs are essential for development and function of inner ear hair cells in vertebrates

MicroRNAs (miRNAs) inhibit the translation of target mRNAs and affect, directly or indirectly, the expression of a large portion of the protein-coding genes. This study focuses on miRNAs that are expressed in the mouse cochlea and vestibule, the 2 inner ear compartments. A conditional knock-out mouse for Dicer1 demonstrated that miRNAs are crucial for postnatal survival of functional hair cells of the inner ear. We identified miRNAs that have a role in the vertebrate developing inner ear by combining miRNA transcriptome analysis, spatial and temporal expression patterns, and bioinformatics. Microarrays revealed similar miRNA profiles in newborn-mouse whole cochleae and vestibules, but different temporal and spatial expression patterns of six miRNAs (miR-15a, miR-18a, miR-30b, miR-99a, miR-182, and miR-199a) may reflect their roles. Two of these miRNAs, miR-15a-1 and miR-18a, were also shown to be crucial for zebrafish inner ear development and morphogenesis. To suggest putative target mRNAs whose translation may be inhibited by selected miRNAs, we combined bioinformatics-based predictions and mRNA expression data. Finally, we present indirect evidence that Slc12a2, Cldn12, and Bdnf mRNAs may be targets for miR-15a. Our data support the hypothesis that inner ear tissue differentiation and maintenance are regulated and controlled by conserved sets of cell-specific miRNAs in both mouse and zebrafish.

Shaping sound in space: the regulation of inner ear patterning

The inner ear is one of the most morphologically elaborate tissues in vertebrates, containing a group of mechanosensitive sensory organs that mediate hearing and balance. These organs are arranged precisely in space and contain intricately patterned sensory epithelia. Here, we review recent studies of inner ear development and patterning which reveal that multiple stages of ear development – ranging from its early induction from the embryonic ectoderm to the establishment of the three cardinal axes and the fine-grained arrangement of sensory cells – are orchestrated by gradients of signaling molecules.

Differential afferent projections to the inferior colliculus from the cochlear nucleus in the albino mouse

The axonal projections from the cochlear nuclear complex to the inferior colliculus (IC) were examined using the retrograde transport of horseradish peroxidase. Thin sheets of neurons in the dorsal and ventral cochlear nuclei were found to project axons in a topographic fashion to restricted laminae of the central nucleus of the IC; the dorsal cochlear nucleus was also found to project axons to the external cortex. No projections were detected from the cochlear nuclear complex to the dorsal cortex of the IC.

Standard atlas of the gross anatomy of the developing inner ear of the chicken.

During development, the chicken inner ear undergoes a series of morphological changes which give rise to the various structures found in the adult, including the mature semicircular canals, utricle, saccule, cochlear duct, endolymphatic duct and sac, and neurons of the eighth cranial nerve ganglion. Beginning as a hollow epithelial sphere, the inner ear is sculpted into this complex labyrinth of fluid‐filled ducts punctuated by their associated sensory end organs. In this report, the three‐dimensional complexity of the developing inner ear of the chicken embryo is documented in the form of a standard atlas. The protocol involved fixation, dehydration, and clearing of embryonic heads harvested at daily intervals, followed by injection of an opaque dye (enamel paint suspension) into the fluid ducts of the inner ear. The position of the ear is shown relative to surface landmarks at seven different stages of development, ranging from embryonic day 5 (E5) to E18. Also shown are higher‐power photomicrographs of the inner ear in isolation taken at daily intervals at E3–E17 and viewed from two orthogonal positions. Three orthogonal views are shown at 6‐hour intervals during the critical stages of semicircular canal formation (E6–E7). Quantitative measurements of the linear dimensions of the inner ear (dorsoventral, anteroposterior, and mediolateral axes) as a function of time indicate a linear increase in the growth of the ear from E3 through E18. This atlas should prove valuable for evaluating mutant phenotypes in inner ear morphogenesis following gene perturbation experiments in the chicken.

Clonal analysis of the relationships between mechanosensory cells and the neurons that innervate them in the chicken ear

In vertebrates, hair-cell-bearing mechanosensory organs and the neurons that innervate them share a common placodal origin. In the inner ear, the peripheral neurons for both auditory and vestibular systems emigrate from the otic placode as neuroblasts, and divide, differentiate and innervate only one of six to eight distinct sensory organs. How these neurons find their correct target is unknown, although one suggestion is that they synapse with clonally related cells. To test this idea for both the middle and inner ears of chicken embryos, lineage analysis was initiated at the time of neuroblast delamination by labeling progenitors with replication-defective retroviruses. The vast majority (89%) of clones were restricted to a single anatomical subdivision of the sensory periphery or its associated ganglia, indicating limited clonal dispersion. Among the remaining clones, we found evidence of a shared neurosensory lineage in the middle ear. Likewise, in the inner ear, neurons could be related to cells of the otic epithelium, although the latter cells were not widely distributed. Rather, they were restricted to a region in or near the utricular macula. None of the other seven sensory organs was related to the ganglion neurons, suggesting that a common lineage between neurons and their targets is not a general mechanism of establishing synaptic connections in the inner ear. This conclusion is further strengthened by finding a shared lineage between the vestibular and acoustic ganglia, revealing the presence of a common progenitor for the two functional classes of neurons.