Methods to study the development, anatomy, and function of the zebrafish inner ear across the life course.

The inner ear is a remarkably intricate structure able to detect sound, motion, and gravity. During development of the zebrafish embryo, the ear undergoes dynamic morphogenesis from a simple epithelial vesicle into a complex labyrinth, consisting of three semicircular canals and three otolithic sensory organs, each with an array of differentiated cell types. This microcosm of biology has led to advances in understanding molecular and cellular changes in epithelial patterning and morphogenesis, through to mechanisms of mechanosensory transduction and the origins of reflexive behavior. In this chapter, we describe different methods to study the zebrafish ear, including high-speed imaging of otic cilia, confocal microscopy, and light-sheet fluorescent microscopy. Many dyes, antibodies, and transgenic lines for labeling the ear are available, and we provide a comprehensive review of these resources. The developing ear is amenable to genetic, chemical, and physical manipulations, including injection and transplantation. Chemical modulation of developmental signaling pathways has paved the way for zebrafish to be widely used in drug discovery. We describe two chemical screens with relevance to the ear: a fluorescent-based screen for compounds that protect against ototoxicity, and an in situ-based screen for modulators of a signaling pathway involved in semicircular canal development. We also describe methods for dissection and imaging of the adult otic epithelia. We review both manual and automated methods to test the function of the inner ear and lateral line, defects in which can lead to altered locomotor behavior. Finally, we review a collection of zebrafish models that are generating new insights into human deafness and vestibular disorders.

Aldosterone decreases glucose-stimulated insulin secretion in vivo in mice and in murine islets.

AIMS/HYPOTHESIS: Aldosterone concentrations increase in obesity and predict the onset of diabetes. We investigated the effects of aldosterone on glucose homeostasis and insulin secretion in vivo and in vitro. METHODS: We assessed insulin sensitivity and insulin secretion in aldosterone synthase-deficient (As [also known as Cyp11b2](-/-)) and wild-type mice using euglycaemic-hyperinsulinaemic and hyperglycaemic clamps, respectively. We also conducted studies during high sodium intake to normalise renin activity and potassium concentration in As (-/-) mice. We subsequently assessed the effect of aldosterone on insulin secretion in vitro in the presence or absence of mineralocorticoid receptor antagonists in isolated C57BL/6J islets and in the MIN6 beta cell line. RESULTS: Fasting glucose concentrations were reduced in As (-/-) mice compared with wild-type. During hyperglycaemic clamps, insulin and C-peptide concentrations increased to a greater extent in As (-/-) than in wild-type mice. This was not attributable to differences in potassium or angiotensin II, as glucose-stimulated insulin secretion was enhanced in As (-/-) mice even during high sodium intake. There was no difference in insulin sensitivity between As (-/-) and wild-type mice in euglycaemic-hyperinsulinaemic clamp studies. In islet and MIN6 beta cell studies, aldosterone inhibited glucose- and isobutylmethylxanthine-stimulated insulin secretion, an effect that was not blocked by mineralocorticoid receptor antagonism, but was prevented by the superoxide dismutase mimetic tempol. CONCLUSIONS/INTERPRETATION: We demonstrated that aldosterone deficiency or excess modulates insulin secretion in vivo and in vitro via reactive oxygen species and in a manner that is independent of mineralocorticoid receptors. These findings provide insight into the mechanism of glucose intolerance in conditions of relative aldosterone excess.

Expression of BMP signalling pathway members in the developing zebrafish inner ear and lateral line.

In this paper we describe the mRNA expression patterns of members of the bone morphogenetic protein (BMP) signalling pathway in the developing zebrafish ear. bmp2b, 4, and 7 are expressed in discrete areas of otic epithelium, some of which correspond to sensory patches. bmp2b and 4 mark the developing cristae before and during the appearance of differentiated hair cells. bmp4 is also expressed in a dorsal, non-sensory region of the ear. Expression of bmps in cristae is conserved between zebrafish, chick, and mouse, but there are also notable differences in ear expression patterns between these species. Of five zebrafish BMP antagonists, only one (follistatin) shows significant expression in the otic epithelium. The type I receptor bmpr-IB shows localised expression in the ear epithelium. Mediators of BMP signalling, smad1 and smad5, are expressed in statoacoustic and lateral line ganglia; smad5 is also expressed at low levels throughout the ear epithelium. An inhibitory smad, smad6, is expressed laterally in the ear epithelium. Lateral line primordia and neuromasts also express bmp2b, 4, follistatin, smad1, and smad5. The conservation of bmp expression in cristae among different species adds weight to the growing evidence that BMPs are required for the development of the vertebrate ear.

Zebrafish sparse corresponds to an orthologue of c-kit and is required for the morphogenesis of a subpopulation of melanocytes, but is not essential for hematopoiesis or primordial germ cell development.

The relative roles of the Kit receptor in promoting the migration and survival of amniote melanocytes are unresolved. We show that, in the zebrafish, Danio rerio, the pigment pattern mutation sparse corresponds to an orthologue of c-kit. This finding allows us to further elucidate morphogenetic roles for this c-kit-related gene in melanocyte morphogenesis. Our analyses of zebrafish melanocyte development demonstrate that the c-kit orthologue identified in this study is required both for normal migration and for survival of embryonic melanocytes. We also find that, in contrast to mouse, the zebrafish c-kit gene that we have identified is not essential for hematopoiesis or primordial germ cell development. These unexpected differences may reflect evolutionary divergence in c-kit functions following gene duplication events in teleosts.

Mutations affecting development of the zebrafish inner ear and lateral line.

Mutations giving rise to anatomical defects in the inner ear have been isolated in a large scale screen for mutations causing visible abnormalities in the zebrafish embryo (Haffter, P., Granato, M., Brand, M. et al. (1996) Development 123, 1-36). 58 mutants have been classified as having a primary ear phenotype; these fall into several phenotypic classes, affecting presence or size of the otoliths, size and shape of the otic vesicle and formation of the semicircular canals, and define at least 20 complementation groups. Mutations in seven genes cause loss of one or both otoliths, but do not appear to affect development of other structures within the ear. Mutations in seven genes affect morphology and patterning of the inner ear epithelium, including formation of the semicircular canals and, in some, development of sensory patches (maculae and cristae). Within this class, dog-eared mutants show abnormal development of semicircular canals and lack cristae within the ear, while in van gogh, semicircular canals fail to form altogether, resulting in a tiny otic vesicle containing a single sensory patch. Both these mutants show defects in the expression of homeobox genes within the otic vesicle. In a further class of mutants, ear size is affected while patterning appears to be relatively normal; mutations in three genes cause expansion of the otic vesicle, while in little ears and microtic, the ear is abnormally small, but still contains all five sensory patches, as in the wild type. Many of the ear and otolith mutants show an expected behavioural phenotype: embryos fail to balance correctly, and may swim on their sides, upside down, or in circles. Several mutants with similar balance defects have also been isolated that have no obvious structural ear defect, but that may include mutants with vestibular dysfunction of the inner ear (Granato, M., van Eeden, F. J. M., Schach, U. et al. (1996) Development, 123, 399-413,). Mutations in 19 genes causing primary defects in other structures also show an ear defect. In particular, ear phenotypes are often found in conjunction with defects of neural crest derivatives (pigment cells and/or cartilaginous elements of the jaw). At least one mutant, dog-eared, shows defects in both the ear and another placodally derived sensory system, the lateral line, while hypersensitive mutants have additional trunk lateral line organs.

Early embryonic expression of XLPOU-60, a Xenopus POU-domain protein.

XLPOU-60 is a Xenopus POU-domain gene whose expression is tightly controlled during early development at transcriptional, post-transcriptional, translational, and post-translational levels. We report the expression pattern of the XLPOU-60 protein; it is first detectable in the stage V oocyte and accumulates rapidly following fertilisation, reaching a peak at the time of the mid-blastula transition. In the blastula, XLPOU-60 protein translated from injected synthetic mRNA enters nuclei. During gastrulation, both transcript and protein are rapidly down-regulated in a cell-autonomous manner; down-regulation is not dependent on cell-cell contact or induction by activin in an animal cap assay. For the mRNA, this down-regulation correlates with changes in the length of its poly(A) tail and is dependent on sequences in the untranslated regions of the transcript. On the basis of its protein expression pattern and known DNA-binding properties, we speculate that XLPOU-60 may play a role in the control of early transcriptional events in the Xenopus embryo.

Nonsense-mediated mRNA decay in Xenopus oocytes and embryos.

Mutant mRNAs carrying a premature stop codon have a reduced half-life in the cells of many species, probably due to the presence of "surveillance" pathways, which selectively target such mRNAs for degradation. It is reported here that this phenomenon may also occur in Xenopus. In vitro-synthesised transcripts encoding a Xenopus POU-domain protein, XLPOU-60, are stable after injection into the oocyte and embryo. However, introduction of a premature stop codon into these transcripts results in their rapid degradation following injection. In contrast, mutant transcripts with additional or deleted codons but retaining a correct reading frame are stable. These results suggest that RNA stability should be considered when designing control mRNAs for Xenopus injection experiments.