Cell cycles and development in the embryonic zebrafish.
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Biomedical subjects
D A Kane
Publications and source records attributed to D A Kane.
Centromeric markers in the zebrafish.
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Animals↗
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.
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Mutations affecting the formation of the notochord in the zebrafish, Danio rerio.
In a large scale screen for mutants with defects in the embryonic development of the zebrafish we identified mutations in four genes,floating head (flh), momo (mom), no tail (ntl), and doc, that are required for early notochord formation. Mutations in flh and ntl have been described previously, while mom and doc are newly identified genes. Mutant mom embryos lack a notochord in the trunk, and trunk somites from the right and left side of the embryo fuse underneath the neural tube. In this respect mom appears similar to flh. In contrast, notochord precursor cells are present in both ntl and doc embryos. In order to gain a greater understanding of the phenotypes, we have analysed the expression of several axial mesoderm markers in mutant embryos of all four genes. In flh and mom, Ntl expression is normal in the germ ring and tailbud, while the expression of Ntl and other notochord markers in the axial mesodermal region is disrupted. Ntl expression is normal in doc embryos until early somitic stages, when there is a reduction in expression which is first seen in anterior regions of the embryo. This suggests a function for doc in the maintenance of ntl expression. Other notochord markers such as twist, sonic hedgehog and axial are not expressed in the axial mesoderm of ntl embryos, their expression parallels the expression of ntl in the axial mesoderm of mutant doc, flh and mom embryos, indicating that ntl is required for the expression of these markers. The role of doc in the expression of the notochord markers appears indirect via ntl. Floor plate formation is disrupted in most regions in flh and mom mutant embryos but is present in mutant ntl and doc embryos. In mutant embryos with strong ntl alleles the band of cells expressing floor plate markers is broadened. A similar broadening is also observed in the axial mesoderm underlying the floor plate of ntl embryos, suggesting a direct involvement of the notochord precursor cells in floor plate induction. Mutations in all of these four genes result in embryos lacking a horizontal myoseptum and muscle pioneer cells, both of which are thought to be induced by the notochord. These somite defects can be traced back to an impairment of the specification of the adaxial cells during early stages of development. Transplantation of wild-type cells into mutant doc embryos reveals that wild-type notochord cells are sufficient to induce horizontal myoseptum formation in the flanking mutant tissue. Thus doc, like flh and ntl, acts cell autonomously in the notochord. In addition to the four mutants with defects in early notochord formation, we have isolated 84 mutants, defining at least 15 genes, with defects in later stages of notochord development. These are listed in an appendix to this study.
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Mutations affecting development of the midline and general body shape during zebrafish embryogenesis.
Tissues of the dorsal midline of vertebrate embryos, such as notochord and floor plate, have been implicated in inductive interactions that pattern the neural tube and somites. In our screen for embryonic visible mutations in the zebrafish we found 113 mutations in more than 27 genes with altered body shape, often with additional defects in CNS development. We concentrated on a subgroup of mutations in ten genes (the midline-group) that cause defective development of the floor plate. By using floor plate markers, such as the signaling molecule sonic hedgehog, we show that the schmalspur (sur) gene is needed for early floor plate development, similar to one-eyed-pinhead (oep) and the previously described cyclops (cyc) gene. In contrast to oep and cyc, sur embryos show deletions of ventral CNS tissue restricted to the mid- and hindbrain, whereas the forebrain appears largely unaffected. In the underlying mesendodermal tissue of the head, sur is needed only for development of the posterior prechordal plate, whereas oep and cyc are required for both anterior and posterior prechordal plate development. Our analysis of sur mutants suggests that defects within the posterior prechordal plate may cause aberrant development of ventral CNS structures in the mid- and hindbrain. Later development of the floor plate is affected in mutant chameleon, you-too, sonic-you, iguana, detour, schmalhans and monorail embryos; these mutants often show additional defects in tissues that are known to depend on signals from notochord and floor plate. For example, sur, con and yot mutants show reduction of motor neurons; median deletions of brain tissue are seen in sur, con and yot embryos; and cyc, con, yot, igu and dtr mutants often show no or abnormal formation of the optic chiasm. We also find fusions of the ventral neurocranium for all midline mutants tested, which may reveal a hitherto unrecognized function of the midline in influencing differentiation of neural crest cells at their destination. As a working hypothesis, we propose that midline-group genes may act to maintain proper structure and inductive function of zebrafish midline tissues.
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Mutations affecting morphogenesis during gastrulation and tail formation in the zebrafish, Danio rerio.
We have identified several genes that are required for various morphogenetic processes during gastrulation and tail formation. Two genes are required in the anterior region of the body axis: one eyed pinhead (oep) and dirty nose (dns).oep mutant embryos are defective in prechordal plate formation and the specification of anterior and ventral structures of the central nervous system. In dns mutants, cells of the prechordal plate, such as the prospective hatching gland cells, fail to specify. Two genes are required for convergence and extension movements. In mutant trilobite embryos, extension movements on the dorsal side of the embryo are affected, whereas in the formerly described spadetail mutants, for which two new alleles have been isolated, convergent movements of ventrolateral cells to the dorsal side are blocked. Two genes are required for the development of the posterior end of the body axis. In pipetail mutants, the tailbud fails to move ventrally on the yolk sac after germ ring closure, and the tip of the tail fails to detach from the yolk tube. Mutants in kugelig (kgg) do not form the yolk tube at the posterior side of the yolk sac.
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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.
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Mutations in zebrafish genes affecting the formation of the boundary between midbrain and hindbrain.
Mutations in two genes affect the formation of the boundary between midbrain and hindbrain (MHB): no isthmus (noi) and acerebellar (ace). noi mutant embryos lack the MHB constriction, the cerebellum and optic tectum, as well as the pronephric duct. Analysis of noi mutant embryos with neuron-specific antibodies shows that the MHB region and the dorsal and ventral midbrain are absent or abnormal, but that the rostral hindbrain is unaffected with the exception of the cerebellum. Using markers that are expressed during its formation (eng, wnt1 and pax-b), we find that the MHB region is already misspecified in noi mutant embryos during late gastrulation. The tectum is initially present and later degenerates. The defect in ace mutant embryos is more restricted: MHB and cerebellum are absent, but a tectum is formed. Molecular organisation of the tectum and tegmentum is disturbed, however, since eng, wnt1 and pax-b marker gene expression is not maintained. We propose that noi and ace are required for development of the MHB region and of the adjacent mid- and hindbrain, which are thought to be patterned by the MHB region. Presence of pax-b RNA, and absence of pax-b protein, together with the observation of genetic linkage and the occurrence of a point mutation, show that noi mutations are located in the pax-b gene. pax-b is a vertebrate orthologue of the Drosophila gene paired, which is involved in a pathway of cellular interactions at the posterior compartment boundary in Drosophila. Our results confirm and extend a previous report, and show that at least one member of this conserved signalling pathway is required for formation of the boundary between midbrain and hindbrain in the zebrafish.
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Genes involved in forebrain development in the zebrafish, Danio rerio.
We identified four zebrafish mutants with defects in forebrain induction and patterning during embryogenesis. The four mutants define three genes: masterblind (mbl), silberblick (slb), and knollnase (kas). In mbl embryos, the anterior forebrain acquires posterior forebrain characteristics: anterior structures such as the eyes, olfactory placodes and the telencephalon are missing, whereas the epiphysis located in the posterior forebrain is expanded. In slb embryos, the extension of the embryonic axis is initially delayed and eventually followed by a partial fusion of the eyes. Finally, in kas embryos, separation of the telencephalic primordia is incomplete and dorsal midline cells fail to form a differentiated roof plate. Analysis of the mutant phenotypes indicates that we have identified genes essential for the specification of the anterior forebrain (mbl), positioning of the eyes (slb) and differentiation of the roof plate (kas). In an appendix to this study we list mutants showing alterations in the size of the eyes and abnormal differentiation of the lenses.
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Mutations affecting neurogenesis and brain morphology in the zebrafish, Danio rerio.
In a screen for embryonic mutants in the zebrafish a large number of mutants were isolated with abnormal brain morphology. We describe here 26 mutants in 13 complementation groups that show abnormal development of large regions of the brain. Early neurogenesis is affected in white tail (wit). During segmentation stages, homozygous wit embryos display an irregularly formed neural keel, particularly in the hindbrain. Using a variety of molecular markers, a severe increase in the number of various early differentiating neurons can be demonstrated. In contrast, late differentiating neurons, radial glial cells and some nonneural cell types, such as the neural crest-derived melanoblasts, are much reduced. Somitogenesis appears delayed. In addition, very reduced numbers of melanophores are present posterior to the mid-trunk. The wit phenotype is reminiscent of neurogenic mutants in Drosophila, such as Notch or Delta. In mutant parachute (pac) embryos the general organization of the hindbrain is disturbed and many rounded cells accumulate loosely in the hindbrain and midbrain ventricles. Mutants in a group of 6 genes, snakehead(snk), natter (nat), otter (ott), fullbrain (ful), viper (vip) and white snake (wis) develop collapsed brain ventricles, before showing signs of general degeneration. atlantis (atl), big head (bid), wicked brain (win), scabland (sbd) and eisspalte (ele) mutants have different malformation of the brain folds. Some of them have transient phenotypes, and mutant individuals may grow up to adults.
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Neural degeneration mutants in the zebrafish, Danio rerio.
Forty zebrafish mutants with localized or general neural degeneration are described. The onset and duration of degeneration and the distribution of ectopically dying cells are specific characteristics of each mutant. Mutants are classified into four groups by these parameters. Class I: late focal neural degeneration mutants. These 18 mutants have restricted cell death mainly in the tectum and the dorsal hindbrain after 36 hours. The degeneration does not spread and disappears at later stages of development. Class II: early focal neural degeneration mutants. Ten mutants in this class exhibit transient restricted degeneration affecting mainly the diencephalon, the hindbrain and the spinal cord at 20 hours. The midbrain is less affected. The degeneration shifts to the dorsal diencephalon and the tectum at 36 hours. Class III: late spreading neural degeneration mutants. The 8 mutants in this class display a degeneration that is first seen in the tectum and subsequently spreads throughout the nervous system from 36 hours on. Class IV: early general neural degeneration mutants. This class of four mutants already shows overall cell degeneration in the nervous system at the 15-somite stage. 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.
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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.
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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.
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Mutations affecting the cardiovascular system and other internal organs in zebrafish.
In a screen for early developmental mutants of the zebrafish, we have identified mutations specifically affecting the internal organs. We identified 53 mutations affecting the cardiovascular system. Nine of them affect specific landmarks of heart morphogenesis. Mutations in four genes cause a failure in the fusion of the bilateral heart primordia, resulting in cardia bifida. In lonely atrium, no heart venticle is visible and the atrium is directly fused to the outflow tract. In the overlooped mutant, the relative position of the two heart chambers is distorted. The heart is enormously enlarged in the santa mutant. In two mutants, scotch tape and superglue, the cardiac jelly between the two layers of the heart is significantly reduced. We also identified a number of mutations affecting the function of the heart. The mutations affecting heart function can be subdivided into two groups, one affecting heart contraction and another affecting the rhythm of the heart beat. Among the contractility group of mutants are 5 with no heart beat at all and 15 with a reduced heart beat of one or both chambers. 6 mutations are in the rhythmicity group and specifically affect the beating pattern of the heart. Mutations in two genes, bypass and kurzschluss, cause specific defects in the circulatory system. In addition to the heart mutants, we identified 23 mutations affecting the integrity of the liver, the intestine or the kidney. In this report, we demonstrate that it is feasible to screen for genes specific for the patterning or function of certain internal organs in the zebrafish. The mutations presented here could serve as an entry point to the establishment of a genetic hierarchy underlying organogenesis.
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Characterization of zebrafish mutants with defects in embryonic hematopoiesis.
As part of a large scale chemical mutagenesis screen of the zebrafish (Danio rerio) genome, we have identified 33 mutants with defects in hematopoiesis. Complementation analysis placed 32 of these mutants into 17 complementation groups. The allelism of the remaining 1 blood mutant is currently unresolved. We have categorized these blood mutants into four phenotypic classes based on analyses of whole embryos and isolated blood cells, as well as by in situ hybridization using the hematopoietic transcription factors GATA-1 and GATA-2. Embryos mutant for the gene moonshine have few if any proerythroblasts visible on the day circulation begins and normal erythroid cell differentiation is blocked as determined by staining for hemoglobin and GATA-1 expression. Mutations in five genes, chablis, frascati, merlot, retsina, thunderbird and two possibly unique mutations cause a progressive decrease in the number of blood cells during the first 5 days of development. Mutations in another seven genes, chardonnay, chianti, grenache, sauternes, weiflherbst and zinfandel, and two additional mutations result in hypochromic blood cells which also decrease in number as development proceeds. Several of these mutants have immature cells in the circulation, indicating a block in normal erythroid development. The mutation in zinfandel is dominant, and 2-day old heterozygous carriers fail to express detectable levels of hemoglobin and have decreasing numbers of circulating cells during the first 5 days of development. Mutations in two genes, freixenet and yquem, result in the animals that are photosensitive with autofluorescent blood, similar to that found in the human congenital porphyrias. The collection of mutants presented here represent several steps required for normal erythropoiesis. The analysis of these mutants provides a powerful approach towards defining the molecular mechanisms involved in vertebrate hematopoietic development.
Anemia, Hypochromic↗
Jaw and branchial arch mutants in zebrafish I: branchial arches.
Jaws and branchial arches together are a basic, segmented feature of the vertebrate head. Seven arches develop in the zebrafish embryo (Danio rerio), derived largely from neural crest cells that form the cartilaginous skeleton. In this and the following paper we describe the phenotypes of 109 arch mutants, focusing here on three classes that affect the posterior pharyngeal arches, including the hyoid and five gill-bearing arches. In lockjaw, the hyoid arch is strongly reduced and subsets of branchial arches do not develop. Mutants of a large second class, designated the flathead group, lack several adjacent branchial arches and their associated cartilages. Five alleles at the flathead locus all lead to larvae that lack arches 4-6. Among 34 other flathead group members complementation tests are incomplete, but at least six unique phenotypes can be distinguished. These all delete continuous stretches of adjacent branchial arches and unpaired cartilages in the ventral midline. Many show cell death in the midbrain, from which some neural crest precursors of the arches originate. lockjaw and a few mutants in the flathead group, including pistachio, affect both jaw cartilage and pigmentation, reflecting essential functions of these genes in at least two neural crest lineages. Mutants of a third class, including boxer, dackel and pincher, affect pectoral fins and axonal trajectories in the brain, as well as the arches. Their skeletal phenotypes suggest that they disrupt cartilage morphogenesis in all arches. Our results suggest that there are sets of genes that: (1) specify neural crest cells in groups of adjacent head segments, and (2) function in common genetic pathways in a variety of tissues including the brain, pectoral fins and pigment cells as well as pharyngeal arches.
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Jaw and branchial arch mutants in zebrafish II: anterior arches and cartilage differentiation.
In a large scale screen for mutants that affect the early development of the zebrafish, 109 mutants were found that cause defects in the formation of the jaw and the more posterior pharyngeal arches. Here we present the phenotypic description and results of the complementation analysis of mutants belonging to two major classes: (1) mutants with defects in the mandibular and hyoid arches and (2) mutants with defects in cartilage differentiation and growth in all arches. Mutations in four of the genes identified during the screen show specific defects in the first two arches and leave the more posterior pharyngeal arches largely unaffected (schmerle, sucker, hoover and sturgeon). In these mutants ventral components of the mandibular and hyoid arches are reduced (Meckel's cartilage and ceratohyal cartilage) whereas dorsal structures (palatoquadrate and hyosymplectic cartilages) are of normal size or enlarged. Thus, mutations in single genes cause defects in the formation of first and second arch structures but also differentially affect development of the dorsal and ventral structures within one arch. In 27 mutants that define at least 8 genes, the differentiation of cartilage and growth is affected. In hammerhead mutants particularly the mesodermally derived cartilages are reduced, whereas jellyfish mutant larvae are characterized by a severe reduction of all cartilaginous elements, leaving only two pieces in the position of the ceratohyal cartilages. In all other mutant larvae all skeletal elements are present, but consist of smaller and disorganized chondrocytes. These mutants also exhibit shortened heads and reduced pectoral fins. In homozygous knorrig embryos, tumor-like outgrowths of chondrocytes occur along the edges of all cartilaginous elements. The mutants presented here may be valuable tools for elucidating the genetic mechanisms that underlie the development of the mandibular and the hyoid arches, as well as the process of cartilage differentiation.
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Zebrafish pigmentation mutations and the processes of neural crest development.
Neural crest development involves cell-fate specification, proliferation, patterned cell migration, survival and differentiation. Zebrafish neural crest derivatives include three distinct chromatophores, which are well-suited to genetic analysis of their development. As part of a large-scale mutagenesis screen for embryonic/early larval mutations, we have isolated 285 mutations affecting all aspects of zebrafish larval pigmentation. By complementation analysis, we define 94 genes. We show here that comparison of their phenotypes permits classification of these mutations according to the types of defects they cause, and these suggest which process of neural crest development is probably affected. Mutations in eight genes affect the number of chromatophores: these include strong candidates for genes necessary for the processes of pigment cell specification and proliferation. Mutations in five genes remove part of the wild-type pigment pattern, and suggest a role in larval pigment pattern formation. Mutations in five genes show ectopic chromatophores in distinct sites, and may have implications for chromatophore patterning and proliferation. 76 genes affect pigment or morphology of one or more chromatophore types: these mutations include strong candidates for genes important in various aspects of chromatophore differentiation and survival. In combination with the embryological advantages of zebrafish, these mutations should permit cellular and molecular dissection of many aspects of neural crest development.
Adaptation, Physiological↗