Mutations causing a visible phenotype in the adult serve as valuable visible genetic markers in multicellular genetic model organisms such as Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana. In a large scale screen for mutations affecting early development of the zebrafish, we identified a number of mutations that are homozygous viable or semiviable. Here we describe viable mutations which produce visible phenotypes in the adult fish. These predominantly affect the fins and pigmentation, but also the eyes and body length of the adult. A number of dominant mutations caused visible phenotypes in the adult fish. Mutations in three genes, long fin, another long fin and wanda affected fin formation in the adult. Four mutations were found to cause a dominant reduction of the overall body length in the adult. The adult pigment pattern was found to be changed by dominant mutations in wanda, asterix, obelix, leopard, salz and pfeffer. Among the recessive mutations producing visible phenotypes in the homozygous adult, a group of mutations that failed to produce melanin was assayed for tyrosinase activity. Mutations in sandy produced embryos that failed to express tyrosinase activity. These are potentially useful for using tyrosinase as a marker for the generation of transgenic lines of zebrafish.

To investigate what role homeotic genes may play in morphological evolution, we are comparing homeotic gene expression in two very different insects, Drosophila (Diptera) and Schistocerca (Orthoptera). In this paper we describe a monoclonal antibody, FP6.87, that recognizes the products of both the Ultrabithorax (Ubx) and abdominal-A (abd-A) genes in Drosophila, via an epitope common to the carboxy terminal region of these two proteins. This antibody recognizes nuclear antigens present in the posterior thorax and abdomen of Schistocerca. We infer that it recognizes the Schistocerca homolog of UBX protein, and probably also of ABD-A. As the distribution of Schistocerca ABD-A protein is already known, we can use this reagent to map the expression of Schistocerca UBX in the thorax and anterior abdomen, where ABD-A is not expressed. Both the general domain, and many of the details, of UBX expression are remarkably conserved compared with Drosophila. Thus UBX expression extends back from T2 in the ectoderm (including the CNS), but only from A1 in the mesoderm. As noted for other bithorax complex genes in Schistocerca, expression begins in the abdomen, at or shortly before the time of segmentation. It only later spreads anteriorly to the thorax. For much of embryogenesis, the expression of UBX in the thoracic epidermis is largely restricted to the T3 limb. In this limb, UBX is strikingly regulated, in a complex pattern that reflects limb segmentation. Reviewing these and earlier observations, we conclude that evolutionary changes affect both the precise regulation of homeotic genes within segments, and probably also the spectrum of downstream genes that respond to homeotic gene expression in a given tissue. Overall domains of homeotic gene expression appear to be well conserved between different insect groups, though a change in the extent and timing of homeotic gene expression may underlie the modification of the posterior abdomen in different insect groups.

Three of the class I mutants show a change in the pattern of gene expression in the anlage of a brain structure prior to the onset of degeneration. These results suggest that focal cell death may be a useful clue for the detection of early patterning defects of the vertebrate nervous system in regions devoid of visible landmarks.

In a large-scale screen, we isolated mutants displaying a specific visible phenotype in embryos or early larvae of the zebrafish, Danio rerio. Males were mutagenized with ethylnitrosourea (ENU) and F2 families of single pair matings between sibling F1 fish, heterozygous for a mutagenized genome, were raised. Egg lays were obtained from several crosses between F2 siblings, resulting in scoring of 3857 mutagenized genomes. F3 progeny were scored at the second, third and sixth day of development, using a stereomicroscope. In a subsequent screen, fixed embryos were analyzed for correct retinotectal projection. A total of 4264 mutants were identified. Two thirds of the mutants displaying rather general abnormalities were eventually discarded. We kept and characterized 1163 mutants. In complementation crosses performed between mutants with similar phenotypes, 894 mutants have been assigned to 372 genes. The average allele frequency is 2.4. We identified genes involved in early development, notochord, brain, spinal cord, somites, muscles, heart, circulation, blood, skin, fin, eye, otic vesicle, jaw and branchial arches, pigment pattern, pigment formation, gut, liver, motility and touch response. Our collection contains alleles of almost all previously described zebrafish mutants. From the allele frequencies and other considerations we estimate that the 372 genes defined by the mutants probably represent more than half of all genes that could have been discovered using the criteria of our screen. Here we give an overview of the spectrum of mutant phenotypes obtained, and discuss the limits and the potentials of a genetic saturation screen in the zebrafish.

Waardenburg-Shah syndrome combines the reduced enteric nervous system characteristic of Hirschsprung’s disease with reduced pigment cell number, although the cell biological basis of the disease is unclear. We have analysed a zebrafish Waardenburg-Shah syndrome model. We show that the colourless gene encodes a sox10 homologue, identify sox10 lesions in mutant alleles and rescue the mutant phenotype by ectopic sox10 expression. Using iontophoretic labelling of neural crest cells, we demonstrate that colourless mutant neural crest cells form ectomesenchymal fates. By contrast, neural crest cells which in wild types form non-ectomesenchymal fates generally fail to migrate and do not overtly differentiate. These cells die by apoptosis between 35 and 45 hours post fertilisation. We provide evidence that melanophore defects in colourless mutants can be largely explained by disruption of nacre/mitf expression. We propose that all defects of affected crest derivatives are consistent with a primary role for colourless/sox10 in specification of non-ectomesenchymal crest derivatives. This suggests a novel mechanism for the aetiology of Waardenburg-Shah syndrome in which affected neural crest derivatives fail to be generated from the neural crest.