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The zebrafish pronephric kidney provides a simplified model of nephron development and epithelial cell differentiation which is amenable to genetic analysis. The pronephros consists of two nephrons with fused glomeruli and paired pronephric tubules and ducts. Nephron formation occurs after the differentiation of the pronephric duct with both the glomeruli and tubules being derived from a nephron primordium. Fluorescent dextran injection experiments demonstrate that vascularization of the zebrafish pronephros and the onset of glomerular filtration occurs between 40 and 48 hpf. We isolated fifteen recessive mutations that affect development of the pronephros. All have visible cysts in place of the pronephric tubule at 2-2.5 days of development. Mutants were grouped in three classes: (1) a group of twelve mutants with defects in body axis curvature and manifesting the most rapid and severe cyst formation involving the glomerulus, tubule and duct, (2) the fleer mutation with distended glomerular capillary loops and cystic tubules, and (3) the mutation pao pao tang with a normal glomerulus and cysts limited to the pronephric tubules. double bubble was analyzed as a representative of mutations that perturb the entire length of the pronephros and body axis curvature. Cyst formation begins in the glomerulus at 40 hpf at the time when glomerular filtration is established suggesting a defect associated with the onset of pronephric function. Basolateral membrane protein targeting in the pronephric duct epithelial cells is also severely affected, suggesting a failure in terminal epithelial cell differentiation and alterations in electrolyte transport. These studies reveal the similarity of normal pronephric development to kidney organogenesis in all vertebrates and allow for a genetic dissection of genes needed to establish the earliest renal function.

In a large-scale screen for mutations affecting embryogenesis in zebrafish, we identified 48 mutations in 34 genetic loci specifically affecting craniofacial development. Mutants were analyzed for abnormalities in the cartilaginous head skeleton. Further, the expression of marker genes was studied to investigate potential abnormalities in mutant rhombencephalon, neural crest, and pharyngeal endoderm. The results suggest that the identified mutations affect three distinct aspects of craniofacial development. In one group, mutations affect the overall pattern of the craniofacial skeleton, suggesting that the genes are involved in the specification of these elements. Another large group of mutations affects differentiation and morphogenesis of cartilage, and may provide insight into the genetic control of chondrogenesis. The last group of mutations leads to the abnormal arrangement of skeletal elements and may uncover important tissue-tissue interactions underlying jaw development.

In a large scale mutagenesis screen for embryonic mutants in zebrafish, we have identified 63 mutations in 24 loci affecting the morphogenesis of the zebrafish brain. The expression of marker genes and the integrity of the axonal scaffold have been studied to investigate abnormalities in regionalization, neurogenesis and axonogenesis in the brain. Mutants can be broadly classified into two groups, one affecting regionalization along the anterior-posterior or dorsal-ventral axis, and the other affecting general features of brain morphology. The first group includes one locus that is required to generate the anlage of the midbrain-hindbrain boundary region at the beginning of somitogenesis. Four loci were identified that affect dorsal-ventral patterning of the brain, including the previously described cyclops locus. Mutant embryos of this class show a reduction of ventral neuroectodermal structures and variable fusion of the eyes. The second group includes a large class of mutations affecting the formation of brain ventricles. Analysis of this class reveals the requirement of a functional cardiovascular system for ventricle enlargement during embryogenesis. Mutations in one locus lead to the formation of supernumerary primary neurons, a phenotype reminiscent of neurogenic mutants in Drosophila. Other mutant phenotypes described here range from abnormalities in the fasciculation and outgrowth of axons to defects in the diameter of the neural tube. The identified loci establish the genetic foundation for a further analysis of the development of the zebrafish embryonic brain.

In a large scale screen for genetic defects in zebrafish embryogenesis we identified mutations affecting several aspects of ear development, including: specification of the otic placode, growth of the otic vesicle (otocyst), otolith formation, morphogenesis of the semicircular canals and differentiation of the otic capsule. Here we report initial phenotypic and genetic characterization of 20 of these mutations defining 13 independent loci. Embryos mutant at the quadro locus display abnormal specification of the otic placode. As revealed by dlx-3 expression, the otic field in the mutant embryos is smaller or split into two fields. At later stages of development the ear of quadro mutants is frequently divided into two smaller, incomplete units. Four loci affect ear shape shortly after formation of the otic vesicle. All of them also display abnormal brain morphology. Mutations in five loci result in the absence of otolith formation; two of these also produce changes of ear morphology. Two loci, little richard and golas, affect morphology of the otic vesicle shortly before formation of the semicircular canals. In both cases the morphogenesis of the semicircular canals is disrupted. Finally, the antytalent locus is involved in late expansion of the ear structure. Analysis of mutations presented here will strengthen our understanding of vertebrate ear morphogenesis and provide novel entry points to its genetic analysis.

In a large scale screen for genetic defects in zebrafish embryogenesis we identified 49 mutations affecting development of the retina. Based on analysis of living embryos as well as histological sections, we grouped the isolated mutations into six phenotypic categories. (1) Mutations in three loci result in a loss of wild-type laminar pattern of the neural retina. (2) Defects in four loci lead to an abnormal specification of the eye anlagen. Only one eye frequently forms in this class of mutants. (3) Seven loci predominantly affect development of the outer retinal layers. Mutants in this category display cell loss mainly in the photoreceptor cell layer. (4) Nine mutations cause retardation of eye growth without any other obvious abnormalities in the retina. (5) A group of twelve mutations is characterized by nonspecific retinal degeneration. (6) Four mutations display retinal degeneration associated with a pigmentation defect. Finally, two mutations, one with absence of the ventral retina and one with an eye-specific pigmentation defect, are not classified in any of the above groups. The identified mutations affect numerous aspects of eye development, including: specification of the eye anlage, growth rate of the optic cup, establishment of retinal stratification, specification or differentiation of retinal neurons and formation of the dorsoventral axis in the developing eye.

The zebrafish gastrointestinal system matures in a manner akin to higher vertebrates. We describe nine mutations that perturb development of these organs. Normally, by the fourth day postfertilization the digestive organs are formed, the epithelial cells of the intestine are polarized and express digestive enzymes, the hepatocytes secrete bile, and the pancreatic islets and acini generate immunoreactive insulin and carboxypeptidase A, respectively. Seven mutations cause arrest of intestinal epithelial development after formation of the tube but before cell polarization is completed. These perturb different regions of the intestine. Six preferentially affect foregut, and one the hindgut. In one of the foregut mutations the esophagus does not form. Two mutations cause hepatic degeneration. The pancreas is affected in four mutants, all of which also perturb anterior intestine. The pancreatic exocrine cells are selectively affected in these four mutations. Exocrine precursor cells appear, as identified by GATA-5 expression, but do not differentiate and acini do not form. The pancreatic islets are spared, and endocrine cells mature and synthesize insulin. These gastrointestinal mutations may be informative with regard to patterning and crucial lineage decisions during organogenesis, and may be relevant to diabetes, congenital dysmorphogenesis and disorders of cell proliferation.

The notochord is critical for the normal development of vertebrate embryos. It serves both as the major skeletal element of the embryo and as a signaling source for the establishment of pattern within the neurectoderm, the paraxial mesoderm and other tissues. In a large-scale systematic screen of mutations affecting embryogenesis in zebrafish we identified 65 mutations that fall into 29 complementation groups, each leading to a defect in the formation and/or maintenance of the notochord. These mutations produce phenotypic abnormalities at numerous stages of notochord development, thereby establishing a phenotypic pathway, which in turn suggests a genetic pathway for the development of the notochord. Perturbations within adjacent tissues in mutant embryos further indicate the importance of notochord-derived signals for patterning within the embryo and suggest that these mutations will yield additional insight into the cues that regulate these patterning processes.

One of the major challenges of developmental biology is understanding the inductive and morphogenetic processes that shape the vertebrate embryo. In a large-scale genetic screen for zygotic effect, embryonic lethal mutations in zebrafish we have identified 25 mutations that affect specification of cell fates and/or cellular rearrangements during gastrulation. These mutations define at least 14 complementation groups, four of which correspond to previously identified genes. Phenotypic analysis of the ten novel loci revealed three groups of mutations causing distinct effects on cell fates in the gastrula. One group comprises mutations that lead to deficiencies in dorsal mesodermal fates and affect central nervous system patterning. Mutations from the second group affect formation of ventroposterior embryonic structures. We suggest that mutations in these two groups identify genes necessary for the formation, maintenance or function of the dorsal organizer and the ventral signaling pathway, respectively. Mutations in the third group affect primarily cellular rearrangements during gastrulation and have complex effects on cell fates in the embryo. This group, and to some extent mutations from the first two groups, affect the major morphogenetic processes, epiboly, convergence and extension, and tail morphogenesis. These mutations provide an approach to understanding the genetic control of gastrulation in vertebrates.

As part of a large-scale mutagenesis screen of the zebrafish genome, we have identified 58 mutations that affect the formation and function of the cardiovascular system. The cardiovascular system is particularly amenable for screening in the transparent zebrafish embryo because the heart and blood vessels are prominent and their function easily examined. We have classified the mutations affecting the heart into those that affect primarily either morphogenesis or function. Nine mutations clearly disrupt the formation of the heart. cloche deletes the endocardium. In cloche mutants, the myocardial layer forms in the absence of the endocardium but is dysmorphic and exhibits a weak contractility. Two loci, miles apart and bonnie and clyde, play a critical role in the fusion of the bilateral tubular primordia. Three mutations lead to an abnormally large heart and one to the formation of a diminutive, dysmorphic heart. We have found no mutation that deletes the myocardial cells altogether, but one, pandora, appears to eliminate the ventricle selectively. Seven mutations interfere with vascular integrity, as indicated by hemorrhage at particular sites. In terms of cardiac function, one large group exhibits a weak beat. In this group, five loci affect both chambers and seven a specific chamber (the atrium or ventricle). For example, the weak atrium mutation exhibits an atrium that becomes silent but has a normally beating ventricle. Seven mutations affect the rhythm of the heart causing, for example, a slow rate, a fibrillating pattern or an apparent block to conduction. In several other mutants, regurgitation of blood flow from ventricle to atrium is the most prominent abnormality, due either to the absence of valves or to poor coordination between the chambers with regard to the timing of contraction. The mutations identified in this screen point to discrete and critical steps in the formation and function of the heart and vasculature.

Programmed cell death is a prominent feature of normal animal development. During neurogenesis, naturally occurring cell death is a mechanism to eliminate neurons that fail to make appropriate connections. To prevent accidental cell death, mechanisms that trigger programmed cell death, as well as the genetic components of the cell death program, are tightly controlled. In a large-scale mutagenesis screen for embryonic lethal mutations in zebrafish Danio rerio we have found 481 mutations with a neural degeneration phenotype. Here, we present 50 mutations that fall into two classes (termed spacehead and fala-like) that are characterized by two main features: first, they appear to affect cell survival primarily within the neuroectodermal lineages during somitogenesis, and second, they show an altered brain morphology at or before 28 hours of development. Evidence for the specificity of cell death within the central nervous system comes from visual inspection of dying cells and analysis of DNA fragmentation, a process associated with apoptotic cell death. In mutants, the level of dying cells is significantly increased in brain and spinal cord. Furthermore, at the end of somitogenesis, the cell count of radial glia and trigeminal neurons is reduced in some mutants of the spacehead class. A variety of neurodegenerative disorders in mouse and humans have been associated with abnormal levels of programmed cell death within the central nervous system. The mutations presented here might provide a genetic framework to aid in the understanding of the etiology of degenerative and physiological disorders within the CNS and the activation of inappropriate programmed cell death.

Systematic genome-wide mutagenesis screens for embryonic phenotypes have been instrumental in the understanding of invertebrate and plant development. Here, we report the results from the first application of such a large-scale genetic screening to vertebrate development. Male zebrafish were mutagenized with N-ethyl N-nitrosourea to induce mutations in spermatogonial cells at an average specific locus rate of one in 651 mutagenized genomes. Mutations were transmitted to the F1 generation, and 2205 F2 families were raised. F3 embryos from sibling crosses within the F2 families were screened for developmental abnormalities. A total of 2337 mutagenized genomes were analyzed, and 2383 mutations resulting in abnormal embryonic and early larval phenotypes were identified. The phenotypes of 695 mutants indicated involvement of the identified loci in specific aspects of embryogenesis. These mutations were maintained for further characterization and were classified into categories according to their phenotypes. The analyses and genetic complementation of mutations from several categories are reported in separate manuscripts. Mutations affecting pigmentation, motility, muscle and body shape have not been extensively analyzed and are listed here. A total of 331 mutations were tested for allelism within their respective categories. This defined 220 genetic loci with on average 1.5 alleles per locus. For about two-thirds of all loci only one allele was isolated. Therefore it is not possible to give a reliable estimate on the degree of saturation reached in our screen; however, the number of genes that can mutate to visible embryonic and early larval phenotypes in zebrafish is expected to be several-fold larger than the one for which we have observed mutant alleles during the screen. This screen demonstrates that mutations affecting a variety of developmental processes can be efficiently recovered from zebrafish.