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Anophthalmia and microphthalmia are important birth defects, but their pathogenesis remains incompletely understood. We studied a patient with severe unilateral microphthalmia who had a 2.7 Mb deletion at chromosome 18q22.1 that was inherited from his mother. In-situ hybridization showed that one of the deleted genes, TMX3, was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TMX3 in 162 patients with anophthalmia or microphthalmia, and found two missense substitutions in unrelated patients: c.116G>A, predicting p.Arg39Gln, in a male with unilateral microphthalmia and retinal coloboma, and c.322G>A, predicting p.Asp108Asn, in a female with unilateral microphthalmia and severe micrognathia. We used two antisense morpholinos targeted against the zebrafish TMX3 orthologue, zgc:110025, to examine the effects of reduced gene expression in eye development. We noted that the morphant larvae resulting from both morpholinos had significantly smaller eye sizes and reduced labeling with islet-1 antibody directed against retinal ganglion cells at 2 days post fertilization. Co-injection of human wild type TMX3 mRNA rescued the small eye phenotype obtained with both morpholinos, whereas co-injection of human TMX3(p.Arg39Gln) mutant mRNA, analogous to the mutation in the patient with microphthalmia and coloboma, did not rescue the small eye phenotype. Our results show that haploinsufficiency for TMX3 results in a small eye phenotype and represents a novel genetic cause of microphthalmia and coloboma. Future experiments to determine if other thioredoxins are important in eye morphogenesis and to clarify the mechanism of function of TMX3 in eye development are warranted.

The centriole is the core structure of centrosome and cilium. Failure to restrict centriole duplication to once per cell cycle has serious consequences and is commonly observed in cancer. Despite its medical importance, the mechanism of centriole formation is poorly understood. Asl was previously reported to be a centrosomal protein essential for centrosome function. Here we identify mecD, a severe loss-of-function allele of the asl gene, and demonstrate that it is required for centriole and cilia formation. Similarly, Cep 152, the Asl ortholog in vertebrates, is essential for cilia formation and its function can be partially rescued by the Drosophila Asl. The study of Asl localization suggests that it is closely associated with the centriole wall, but is not part of the centriole structure. By analyzing the biogenesis of centrosomes in cells depleted of Asl, we found that, while pericentriolar material (PCM) function is mildly affected, Asl is essential for daughter centriole formation. The clear absence of several centriolar markers in mecD mutants suggests that Asl is critical early in centriole duplication.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.12.056 Byline: Zac Pujic (a), Yoshihiro Omori (a), Motokazu Tsujikawa (a), Bernard Thisse (b), Christine Thisse (b), Jarema Malicki (a) Keywords: Zebrafish; Eye; Retina; Ganglion; Amacrine; Neurogenesis; sox2 Abstract: To gain an understanding of molecular events that underlie pattern formation in the retina, we evaluated the expression profiles of over 8000 transcripts randomly selected from an embryonic zebrafish library. Detailed analysis of cDNAs that display restricted expression patterns revealed factors that are specifically expressed in single cell classes and are potential regulators of neurogenesis. These cDNAs belong to numerous molecular categories and include cell surface receptors, cytoplasmic enzymes, and transcription factors. To test whether expression patterns that we have uncovered using this approach are indicative of function in neurogenesis, we used morpholino-mediated knockdown approach. The knockdown of soxp, a transcript expressed in the vicinity of the inner plexiform layer, revealed its role in cell type composition of amacrine and ganglion cell layers. Blocking the function of cxcr4b, a chemokine receptor specifically expressed in ganglion cells, suggests a role in ganglion cell survival. These experiments demonstrate that in situ hybridization-based reverse genetic screens can be applied to isolate genetic regulators of neurogenesis. This approach very well complements forward genetic mutagenesis studies previously used to study retinal neurogenesis in zebrafish. Author Affiliation: (a) Department of Ophthalmology, Harvard Medical School, MEEI, r513, 243 Charles Street, Boston, MA 02114, USA (b) Institut de Genetique et de Biologie Moleculaire et Cellulaire, UMR 7104, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP10142, CU de Strasbourg, 67404 Illkirch Cedex, France Article History: Received 16 March 2005; Revised 8 December 2005; Accepted 12 December 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.06.009 Byline: Andrei Avanesov (a), Ralf Dahm (b), The Tuebingen 2000 Screening Consortium, William F. Sewell (c), Jarema J. Malicki (a) Keywords: Zebrafish; Retina; Electroretinogram; Amacrine neuron; Degeneration; Cell fate; Cell death; Neurotrophins Abstract: Amacrine neurons are among the most diverse cell classes in the vertebrate retina. To gain insight into mechanisms vital to the production and survival of amacrine cell types, we investigated a group of mutations in three zebrafish loci: kleks (kle), chiorny (chy), and bergmann (bgm). Mutants of all three genes display a severe loss of selected amacrine cell subpopulations. The numbers of GABA-expressing amacrine interneurons are sharply reduced in all three mutants, while cell loss in other amacrine cell subpopulations varies and some cells are not affected at all. To investigate how amacrine cell loss affects retinal function, we performed electroretinograms on mutant animals. While the kle mutation mostly influences the function of the inner nuclear layer, unexpectedly the chy mutant phenotype also involves a loss of photoreceptor cell activity. The precise ratios and arrangement of amacrine cell subpopulations suggest that cell-cell interactions are involved in the differentiation of this cell class. To test whether defects of such interactions may be, at least in part, responsible for mutant phenotypes, we performed mosaic analysis and demonstrated that the loss of parvalbumin-positive amacrine cells in chy mutants is due to extrinsic (cell-nonautonomous) causes. The phenotype of another amacrine cell subpopulation, the GABA-positive cells, does not display a clear cell-nonautonomy in chy animals. These results indicate that environmental factors, possibly interactions among different subpopulations of amacrine neurons, are involved in the development of the amacrine cell class. Author Affiliation: (a) Department of Ophthalmology, Harvard Medical School/MEEI, 243 Charles Street, Boston, MA 02114, USA (b) Max Planck Institut fur Entwicklungsbiologie, Abteilung III/Genetik, Spemannstrasse 35, 72076 Tubingen, Germany (c) Department of Otolaryngology, Harvard Medical School/MEEI, 243 Charles Street, Boston, MA 02114, USA Article History: Received 7 October 2004; Revised 25 May 2005; Accepted 8 June 2005

Genetic screens in zebrafish identified several loci that play essential roles in the patterning of retinal architecture. Here, we show that one of them, glass onion, encodes the N-cadherin gene. The [glo.sup.m117] mutant allele contains a substitution of the Trp2 residue known for its essential role in the adhesive properties of classic cadherins. Both the [glo.sup.m117] and [pac.sup.tm101b] mutant N-cadherin alleles affect the polarity of the retinal neuroepithelial sheet and, unexpectedly, both result in cell-nonautonomous phenotypes in retinal patterning. The late onset of mutant N-cadherin phenotypes may be due to the ability of classic cadherins to substitute each other's function. Keywords: Zebrafish; Eye; Retina; Cadherin; Neuroepithelium; Adhesion

Abstract Background The size of the vertebrate eye and the retina is likely to be controlled at several stages of embryogenesis by mechanisms that affect cell cycle length as well as cell survival. A mutation in the zebrafish out of sight (out) locus results in a particularly severe reduction of eye size. The goal of this study is to characterize the outm233 mutant, and to determine whether mutations in the out gene cause microphthalmia in humans. Results In this study, we show that the severe reduction of eye size in the outm233 mutant is caused by a mutation in the zebrafish gdf6a gene. Despite the small eye size, the overall retinal architecture appears largely intact, and immunohistochemical studies confirm that all major cell types are present in outm233 retinae. Subtle cell fate and patterning changes are present predominantly in amacrine interneurons. Acridine orange and TUNEL staining reveal that the levels of apoptosis are abnormally high in outm233 mutant eyes during early neurogenesis. Mutation analysis of the GDF6 gene in 200 patients with microphthalmia revealed amino acid substitutions in four of them. In two patients additional skeletal defects were observed. Conclusions This study confirms the essential role of GDF6 in the regulation of vertebrate eye size. The reduced eye size in the zebrafish outm233 mutant is likely to be caused by a transient wave of apoptosis at the onset of neurogenesis. Amino acid substitutions in GDF6 were detected in 4 (2%) of 200 patients with microphthalmia. In two patients different skeletal defects were also observed, suggesting pleitrophic effects of GDF6 variants. Parents carrying these variants are asymptomatic, suggesting that GDF6 sequence alterations are likely to contribute to the phenotype, but are not the sole cause of the disease. Variable expressivity and penetrance suggest a complex non-Mendelian inheritance pattern where other genetic factors may influence the outcome of the phenotype.

Mutation of the glass onion locus causes drastic neuronal patterning defects in the zebrafish retina and brain. The precise stratified appearance of the wild-type retina is absent in the mutants. The glass onion phenotype is first visible shortly after the formation of optic primordia and is characterized by the rounding of cells and disruption of the ventricular surface in the eye and brain neuroepithelia. With exception of the dorsal- and ventral-most regions of the brain, neuroepithelial cells lose their integrity and begin to distribute ectopically. At later stages, the laminar patterning of retinal neurons is severely disrupted. Despite the lack of lamination, individual retinal cell classes differentiate in the glass onion retina. Mosaic analysis reveals that the glass onion mutation acts cell nonautonomously within the retina and brain, as neuroepithelial cell morphology and polarity in these tissues are normal when mutant cells develop in wild-type hosts. We conclude that the glass onion mutation affects cell--cell signaling event(s) involved in the maintenance of the neuroepithelial cell layer shortly after its formation. The disruption of neuroepithelial integrity may be the cause of the neuronal patterning defects following neurogenesis. In addition, the expression of the glass onion phenotype in a subset of neuroepithelial cells as well as its onset following the initial formation of the neuroepithelial sheets indicate the presence of genetically distinct temporal and spatial subdivisions in the development of this histologically uniform tissue. Key Words: eye; mutant; glo; patterning; neuroepithelium.

The seven major cell classes of the vertebrate retina are organized with remarkable precision into distinct layers. The appearance of this architecture during embryogenesis raises two questions of general importance. How do individual cell classes acquire their specialized structures and functions if they all originate from a morphologically uniform cell population? What mechanisms are responsible for the formation of such a complex and exact pattern? Recent advances present an opportunity to apply the tools of forward genetic analysis to identify mutations that affect these mechanisms in zebrafish. Molecular characterization will follow, providing insight into the basis of neuronal patterning in the vertebrate CNS. Trends Neurosci. (2000) 23, 531-541