The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio.

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.

Mutations affecting somite formation and patterning in the zebrafish, Danio rerio.

Somitogenesis is the basis of segmentation of the mesoderm in the trunk and tail of vertebrate embryos. Two groups of mutants with defects in this patterning process have been isolated in our screen for zygotic mutations affecting the embryonic development of the zebrafish (Danio rerio). In mutants of the first group, boundaries between individual somites are invisible early on, although the paraxial mesoderm is present. Later, irregular boundaries between somites are present. Mutations in fused somites (fss) and beamter (bea) affect all somites, whereas mutations in deadly seven (des), after eight (aei) and white tail (wit) only affect the more posterior somites. Mutants of all genes but wit are homozygous viable and fertile. Skeletal stainings and the expression pattern of myoD and snail1 suggest that anteroposterior patterning within individual somites is abnormal. In the second group of mutants, formation of the horizontal myoseptum, which separates the dorsal and ventral part of the myotome, is reduced. Six genes have been defined in this group (you-type genes). you-too mutants show the most severe phenotype; in these the adaxial cells, muscle pioneers and the primary motoneurons are affected, in addition to the horizontal myoseptum. The horizontal myoseptum is also missing in mutants that lack a notochord. The similarity of the somite phenotype in mutants lacking the notochord and in the you-type mutants suggests that the genes mutated in these two groups are involved in a signaling pathway from the notochord, important for patterning of the somites.

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.

Genetic analysis of fin formation in the zebrafish, Danio rerio.

In the zebrafish, Danio rerio, a caudal and pectoral fin fold develop during embryogenesis. At larval stages the caudal fin fold is replaced by four different fins, the unpaired anal, dorsal and tail fins. In addition the paired pelvic fins are formed. We have identified a total of 118 mutations affecting larval fin formation. Mutations in 11 genes lead to abnormal morphology or degeneration of both caudal and pectoral fin folds. Most mutants survive to adulthood and form a surprisingly normal complement of adult fins. Mutations in nine genes result in an increased or reduced size of the pectoral fins. Interestingly, in mutants of one of these genes, dackel (dak), pectoral fin buds form initially, but later the fin epithelium fails to expand. Expression of sonic hedgehog mRNA in the posterior mesenchyme of the pectoral fin bud is initiated in dak embryos, but not maintained. Mutations in five other genes affect adult fin but not larval fin development. Two mutants, longfin (lof) and another longfin (alf) have generally longer fins. Stein und bein (sub) has reduced dorsal and pelvic fins, whereas finless (fls) and wanda (wan) mutants affect all adult fins. Finally, mutations in four genes causing defects in embryonic skin formation will be briefly reported.

Genes controlling and mediating locomotion behavior of the zebrafish embryo and larva.

Zebrafish embryos and larvae have stage-specific patterns of motility or locomotion. Two embryonic structures accomplish this behavior: the central nervous system (CNS) and skeletal muscles. To identify genes that are functionally involved in mediating and controlling different patterns of embryonic and larval motility, we included a simple touch response test in our zebrafish large-scale genetic screen. In total we identified 166 mutants with specific defects in embryonic motility. These mutants fall into 14 phenotypically distinct groups comprising at least 48 genes. Here we describe the various phenotypic groups including mutants with no or reduced motility, mechanosensory defective mutants, 'spastic' mutants, circling mutants and motor circuit defective mutants. In 63 mutants, defining 18 genes, striation of somitic muscles is reduced. Phenotypic analysis provides evidence that these 18 genes have distinct and consecutive functions during somitic muscle development. The genes sloth (slo) and frozen (fro) already act during myoblast differentiation, while 13 genes appear to function later, in the formation of myofibers and the organization of sarcomeres. Mutations in four other genes result in muscle-specific degeneration. 103 mutations, defining at least 30 genes, cause no obvious defects in muscle formation and may instead affect neuronal development. Analysis of the behavioral defects suggests that these genes participate in the diverse locomotion patterns observed, such as touch response, rhythmic tail movements, equilibrium control, or that they simply confer general motility to the animal. In some of these mutants specific defects in the developing nervous system are detected. Mutations in two genes, nevermind (nev) and macho (mao), affect axonal projection in the optic tectum, whereas axon formation and elongation of motorneurons are disrupted by mutations in the diwanka (diw) and the unplugged (unp) genes.