Novel dominant-negative mutation within the six domain of the conserved eye specification gene sine oculis inhibits eye development in Drosophila.

The development of the compound eye of Drosophila is controlled, in part, by the concerted actions of several nuclear proteins that form an intricate regulatory system. One member of this network is sine oculis (so), the founding member of the Six gene family. Mutations within so affect the entire visual system, including the compound eye. The vertebrate homologs Six3 and Six6 also appear to play crucial roles in retinal formation. Mutations in Six3 inhibit retinal formation in chickens and fish, whereas those in Six6 are the underlying cause of bilateral anophthalmia in humans. Together, these phenotypes suggest a conserved role for the Six genes in eye development. In this report, we describe the effects of a dominant-negative mutation of sine oculis on the development of the compound eye of Drosophila. The mutation resides within the Six domain and may have implications for eye development and disease.     

The transmembrane inner ear (tmie) gene contributes to vestibular and lateral line development and function in the zebrafish (Danio rerio)

The inner ear is a complex organ containing sensory tissue, including hair cells, the development of which is not well understood. Our long-term goal is to discover genes critical for the correct formation and function of the inner ear and its sensory tissue. A novel gene, transmembrane inner ear (Tmie), was found to cause hearing-related disorders when defective in mice and humans. A homologous tmie gene in zebrafish was cloned and its expression characterized between 24 and 51 hours post-fertilization. Embryos injected with morpholinos (MO) directed against tmie exhibited circling swimming behavior (approximately 37%), phenocopying mice with Tmie mutations; semicircular canal formation was disrupted, hair cell numbers were reduced, and maturation of electrically active lateral line neuromasts was delayed. As in the mouse, tmie appears to be required for inner ear development and function in the zebrafish and for hair cell maturation in the vestibular and lateral line systems as well.     

Electron microscopical evidence for common inner ear and lateral line efferents in urodeles

The efferents of the lateral line system and the inner ear were examined in urodeles using retrograde labelling with horseradish peroxidase (HRP). The Golgi-like filling thus achieved allowed tracing of the axons of efferent cells from one inner ear to the other and from the lateral line nerves into both inner ears. Electron microscopic examination of the inner ear revealed HRP label only in vesicle filled terminals on hair cells traditionally considered as efferent synapses. These data confirm earlier claims of common bilateral inner ear and lateral line efferents. In addition, the efferent nature of retrogradely labelled rhombencephalic cells is proven by their continuity with ultrastructurally identified efferent synapses in the inner ear.     

Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation

We have identified a novel vertebrate homolog of the Drosophila gene dachshund, Dachshund2 (Dach2). Dach2 is expressed in the developing somite prior to any myogenic genes with an expression profile similar to Pax3, a gene previously shown to induce muscle differentiation. Pax3 and Dach2 participate in a positive regulatory feedback loop, analogous to a feedback loop that exists in Drosophila between the Pax gene eyeless (a Pax6 homolog) and the Drosophila dachshund gene. Although Dach2 alone is unable to induce myogenesis, Dach2 can synergize with Eya2 (a vertebrate homolog of the Drosophila gene eyes absent) to regulate myogenic differentiation. Moreover, Eya2 can also synergize with Six1 (a vertebrate homolog of the Drosophila gene sine oculis) to regulate myogenesis. This synergistic regulation of muscle development by Dach2 with Eya2 and Eya2 with Six1 parallels the synergistic regulation of Drosophila eye formation by dachshund with eyes absent and eyes absent with sine oculis. This synergistic regulation is explained by direct physical interactions between Dach2 and Eya2, and Eya2 and Six1 proteins, analogous to interactions observed between the Drosophila proteins. This study reveals a new layer of regulation in the process of myogenic specification in the somites. Moreover, we show that the Pax, Dach, Eya, and Six genetic network has been conserved across species. However, this genetic network has been used in a novel developmental context, myogenesis rather than eye development, and has been expanded to include gene family members that are not directly homologous, for example Pax3 instead of Pax6.     

Central organization of the efferent supply to the labyrinthine and lateral line receptors of the dogfish

Neurons that provide the efferent innervation of the inner ear and lateral line were located in the brain by applying horseradish peroxidase to appropriate cranial nerves. The efferent neurons are found in a rhombencephalic nucleus, called here the octavolateralis efferent nucleus, which lies at the rostral pole of the visceromotor column. Up to 40% of these neurons are contralateral. The location of efferent cells is not topographically related to the sense organs they innervate. Their axons leave the brain together with other motor axons in the facial and glossopharyngeal nerves and there is evidence that some neurons innervate both the ear and the lateral line. The efferent nucleus receives direct sensory input from the labyrinth, but not from the lateral line. The organization of the efferent neurons and the distribution of their axons indicates that their effect on the sense organs is probably widespread and nonspecific.     

Six3 overexpression initiates the formation of ectopic retina

The homeobox gene sine oculis (so) is essential for visual system formation in Drosophila. A vertebrate member of the so/Six gene family, Six3, is expressed in the developing eye and forebrain. Injection of Six3 RNA into medaka fish embryos causes ectopic Pax6 and Rx2 expression in midbrain and cerebellum, resulting in the formation of ectopic retinal primordia. Injected mouse Six3 RNA initiates ectopic expression of endogenous medaka Six3, uncovering a feedback control of Six3 expression. Initiation of ectopic retina formation reveals a pivotal role for Six3 in vertebrate retina development and hints at a conserved regulatory network underlying vertebrate and invertebrate eye development.     

Postembryonic development of the cranial lateral line canals and neuromasts in zebrafish.

The development of the cranial lateral line canals and neuromast organs are described in postembryonic zebrafish (0-80 days postfertilization). Cranial canal development commences several weeks after hatch, is initiated in the vicinity of individual neuromasts, and occurs in four discrete stages that are described histologically. Neuromasts remain in open canal grooves for several weeks during which they dramatically change shape and increase in size by adding hair cells at a rate one-tenth that in the zebrafish inner ear. Scanning electron microscopy demonstrates that neuromasts elongate perpendicular to the canal axis and the axis of hair cell polarization and that they lack a prominent nonsensory cell population surrounding the hair cells-features that make zebrafish neuromasts unusual among fishes. These results demand a reassessment of neuromast and lateral line canal diversity among fishes and highlight the utility of the lateral line system of postembryonic zebrafish for experimental and genetic studies of the development and growth of hair cell epithelia.     

Muscle-specific expression of the smyd1 gene is controlled by its 5.3-kb promoter and 5'-flanking sequence in zebrafish embryos.

Zebrafish SmyD1 is a SET and MYND domain-containing protein that plays an important role in myofiber maturation and muscle contraction. SmyD1 is required for myofibril organization and sarcomere assembly during myofiber maturation. Whole-mount in situ hybridization revealed that smyd1 mRNAs are specifically expressed in skeletal and cardiac muscles in zebrafish embryos. However, it is unknown if smyd1 is expressed in other striated muscles, such as cranial and fin muscles, and moreover, the regulatory elements required for its muscle-specific expression. We report here the analyses of smyd1 expression using smyd1-gfp transgenic zebrafish. smyd1-gfp transgenic zebrafish were generated using the 5.3-kb smyd1 promoter and its 5'-flanking sequence. GFP expression was found in the skeletal and cardiac muscles of smyd1-gfp transgenic embryos. GFP expression appeared stronger in slow muscles than fast muscles in transgenic zebrafish larvae. In addition, GFP expression was also detected in cranial and fin muscles of smyd1-gfp transgenic zebrafish larvae. In situ hybridization confirmed smyd1 mRNA expression in these tissues, suggesting that the expression of the smyd1-gfp transgene recapitulated that of the endogenous smyd1 gene. Deletion analysis revealed that the 0.5-kb sequence in the proximal promoter of smyd1 was essential for its muscle specificity. Together, these data indicate that smyd1 is specifically expressed in most, if not all, striated muscles, and the muscle specificity is controlled by the 5.3-kb promoter and flanking sequences.     

Six3 promotes the formation of ectopic optic vesicle-like structures in mouse embryos.

A few years ago, three novel murine homeobox genes closely related to the Drosophila sine oculis (so) gene (Six1-3) were isolated and were all included in the Six/so gene family. Because of its early expression in the developing eye field, Six3 was initially thought to be the functional ortholog of the Drosophila so gene. This hypothesis was further supported by the demonstration that ectopic Six3 expression in medaka fish (Oryzias latipes) promotes the formation of ectopic lens and retina tissue. Here, we show that similar to Drosophila, where the eyeless/Pax6 gene regulates the eye-specific expression of so, Six3 expression in the murine lens placodal ectoderm is also controlled by Pax6. We also show that ectopic Six3 expression promotes the formation of ectopic optic vesicle-like structures in the hindbrain-midbrain region of developing mouse embryos.     

Isolation of human ear specific cDNAs and construction of cDNA libraries from surgically removed small amounts of inner ear tissues

We have used representational difference analysis (RDA) for subtractive hybridization of oligo dT primed directionally cloned cDNA libraries from human inner ear tissue and a B-lymphoblast cell line. Two rounds of subtraction-amplification, followed, by differential hybridization of selected clones led to the isolation of genes which were specific to the ear. Sequence analysis of randomly chosen clones revealed the presence of a histidine rich Ca2+binding protein, human dynamin, collagen type 1A1, collagen type 2A1, SPARC, human growth hormone, and several specific genes which had no sequence homology in the data base. Furthermore, to apply these techniques for isolating genes specific to distinct inner ear structures and/or cell types of inner ear for which the starting tissue material is limiting, we have used a modified PCR based protocol to construct representative cDNA libraries. We have characterized a cDNA library constructed from small amounts of inner ear tissues recovered by ablative surgical procedure involving labyrinthectomy. The potential application of these protocols for isolating genes involved in hearing and deafness is discussed.     

Expression of ZIC genes in the development of the chick inner ear and nervous system.

ZIC genes, vertebrate homologues of the Drosophila pair-rule gene odd-paired (opa), function in embryonic pattern formation, in the early stages of central nervous system neurogenesis and in cerebellar maturation. Mouse Zic genes are expressed in restricted, and in some cases overlapping, patterns during development, particularly in the central and peripheral nervous systems. We identified chick ZIC2 in a differential display analysis of the auditory system designed to find genes up-regulated after noise trauma. In this study, we examined the expression of chick ZIC1, ZIC2, and ZIC3 by in situ hybridization in normal inner ear development and in the tissues that influence its development, including the hindbrain, the neural crest, and the periotic mesenchyme. Between Hamburger and Hamilton stages 13 and 24, all three ZIC genes were found in the dorsal periotic mesenchyme adjacent to the developing inner ear. ZIC1 mRNA was expressed in the otocyst epithelium between stages 12 and 24, in some sensory tissue, as well as in a striped pattern in the floorplate of the hindbrain that appears to be complementary to that of Chordin, a gene known to regulate ZIC expression in frogs. Chick ZIC genes are also expressed in the neuroectoderm, paraxial mesenchyme, brain, spinal cord, neural crest, and/or the overlying ectoderm as well as the limb buds. In general, ZIC1 and ZIC2 expression patterns overlapped, although ZIC2 expression was less robust; ZIC3 expression was minimal. These observations suggest that ZIC genes, in addition to their known roles in brain development, may play an important role in the development of the chick inner ear.     

Specification of the mammalian cochlea is dependent on Sonic hedgehog

Organization of the inner ear into auditory and vestibular components is dependent on localized patterns of gene expression within the otic vesicle. Surrounding tissues are known to influence compartmentalization of the otic vesicle, yet the participating signals remain unclear. This study identifies Sonic hedgehog (Shh) secreted by the notochord and/or floor plate as a primary regulator of auditory cell fates within the mouse inner ear. Whereas otic induction proceeds normally in Shh(-/-) embryos, morphogenesis of the inner ear is greatly perturbed by midgestation. Ventral otic derivatives including the cochlear duct and cochleovestibular ganglia failed to develop in the absence of Shh. The origin of the inner ear defects in Shh(-/-) embryos could be traced back to alterations in the expression of a number of genes involved in cell fate specification including Pax2, Otx1, Otx2, Tbx1, and Ngn1. We further show that several of these genes are targets of Shh signaling given their ectopic activation in transgenic mice that misexpress Shh in the inner ear. Taken together, our data support a model whereby auditory cell fates in the otic vesicle are established by the direct action of Shh.     

Multiple mutations in mouse Chd7 provide models for CHARGE syndrome

Mouse ENU mutagenesis programmes have yielded a series of independent mutations on proximal chromosome 4 leading to dominant head-bobbing and circling behaviour due to truncations of the lateral semicircular canal of the inner ear. Here, we report the identification of mutations in the Chd7 gene in nine of these mutant alleles including six nonsense and three splice site mutations. The human CHD7 gene is known to be involved in CHARGE syndrome, which also shows inner ear malformations and a variety of other features with varying penetrance and appears to be due to frequent de novo mutation. We found widespread expression of Chd7 in early development of the mouse in organs affected in CHARGE syndrome including eye, olfactory epithelium, inner ear and vascular system. Closer inspection of heterozygous mutant mice revealed a range of defects with reduced penetrance, such as cleft palate, choanal atresia, septal defects of the heart, haemorrhages, prenatal death, vulva and clitoral defects and keratoconjunctivitis sicca. Many of these defects mimic the features of CHARGE syndrome. There were no obvious features of the gene that might make it more mutable than other genes. We conclude that the large number of mouse mutants and human de novo mutations may be due to the combination of the Chd7 gene being a large target and the fact that many heterozygous carriers of the mutations are viable individuals with a readily detectable phenotype.     

Additive gene actions on the fiber number in the anterior optic tract of Drosophila melanogaster.

The influence of mutations in seven neurological genes on the number of fibers in the anterior optic tract (AOT) of Drosophila melanogaster has been investigated. It is shown that the number of fibers in the AOT can be drastically reduced in single and especially in multiple mutants. However, no evidence for synergistic interactions between the sample of mutations used in the sine oculis (so), reduced optic lobes (rol), minibrain (mnb), and small optic lobes (sol) genes was obtained at the level of the AOT. The rolKS222 and so mutations eliminate similar fiber sets in the AOT, which are distinctly different from those eliminated by solKS58 and mnb1.     

Foxg1 is required for proper separation and formation of sensory cristae during inner ear development

The vestibular portion of the inner ear, the three semicircular canals and their sensory cristae, is responsible for detecting angular head movements. It was proposed that sensory cristae induce formation of their non‐sensory components, the semicircular canals. Here, we analyzed the inner ears of Foxg1^?/? mouse mutants, which display vestibular defects that are in conflict with the above model. In Foxg1^?/? ears, the lateral canal is present without the lateral ampulla, which houses the lateral crista. Our gene expression analyses indicate that at the time when canal specification is thought to occur, the prospective lateral crista is present, which could have induced lateral canal formation prior to its demise. Our genetic fate‐mapping analyses indicate an improper separation between anterior and lateral cristae in Foxg1^?/? mutants. Our data further suggest that a function of Foxg1 in the inner ear is to restrict sensory fate. Developmental Dynamics 238:2725–2734, 2009. © 2009 Wiley‐Liss, Inc.