Studies of a spontaneous lethal mutation at the albino locus in SELH/Bc mice.

We report a new mutation at the albino locus in SELH/Bc mice. The mutation arose spontaneously in a male mouse that appeared to be a somatic and germ line mosaic for a new albino (c) allele, provisionally named cBc. The mutation is a recessive lethal, causing embryonic death soon after implantation. We have shown that there is no detectable activity of the Mod-2 allele in cis with the mutation and conclude that the mutation is probably a deletion that includes the c locus, the Mod-2 locus, the intervening 2 cM, and at least one locus essential for postimplantation embryonic survival, either proximal to the c locus or distal to the Mod-2 locus. This new mutation is similar to most previously reported spontaneous mutations at the albino locus in that it arose in a somatic and germ line mosaic mutant animal but differs from them in that it is an embryonic lethal when homozygous and is apparently a deletion. SELH/Bc mice appear to have a high mutation rate. This lethal albino mutation that appears to be a postmeiotic deletion should be useful in the search for the mechanism of mutagenesis in SELH/Bc mice. It may also be useful in mapping essential genes in the c-locus region.     

ENU-induced allele of brachyury (Tkt1) exhibits a developmental lethal phenotype similar to the original brachyury (T) mutation

New alleles of brachyury (Tkt1, Tkt4) were induced in the mouse complete tw5 haplotype by ethylnitrosourea (ENU). Like the original brachyury (T) mutation, the new alleles cause a short-tailed phenotype in heterozygotes, and interact with the t complex tail interaction factor (tct) in trans to cause phenotypically tailless mice. Because ENU is mainly a point mutagen, it is important to determine that the new alleles are homozygous embryonic lethal mutations like the original T allele, and to characterize their embryonic lethal phenotype. Moreover, the Tkt1 mutation maps to an inverted position relative to quaking (qk) in t haplotypes as compared with its position on normal chromosome 17. The Tkt1 allele was separated from the resident tw5 lethal gene, tclw5, by recombination, allowing embryology studies to be performed. Embryological analyses show that the Tkt1 allele is nearly identical to the classic T allele. At 9 and 10 days of development, homozygous Tkt1/Tkt1 embryos are grossly abnormal with properties including 1) irregular, disorganized somite pairs, 2) a shortened posterior end of the embryo, 3) an irregular neural tube, and 4) an abnormal notochord. In addition, 10 day-old abnormal embryos have anterior limb buds that point dorsally rather than ventrally, and are smaller than normal littermates. We conclude that the Tkt1 mutation is a valuable allele for both mapping and molecular characterization of the brachyury locus.     

Latheo, a New Gene Involved in Associative Learning and Memory in Drosophila Melanogaster, Identified from P Element Mutagenesis

Genetic dissection of learning and memory in Drosophila has been limited by the existence of ethyl methanesulfonate (EMS)-induced mutations in only a small number of X-linked genes. To remedy this shortcoming, we have begun a P element mutagenesis to screen for autosomal mutations that disrupt associative learning and/or memory. The generation of "P-tagged" mutant alleles will expedite molecular cloning of these new genes. Here, we describe a behavior-genetic characterization of latheoP1, a recessive, hypomorphic mutation of an essential gene. latheoP1 flies perform poorly in olfactory avoidance conditioning experiments. This performance deficit could not be attributed to abnormal olfactory acuity or shock reactivity-two task-relevant "peripheral" behaviors which are used during classical conditioning. Thus, the latheoP1 mutation appears to affect learning/memory specifically. Consistent with chromosomal in situ localization of the P element insertion, deficiencies of the 49F region of the second chromosome failed to complement the behavioral effect of the latheoP1 mutation. Further complementation analyses between latheoP1 and lethal alleles, produced by excision of the latheoP1 insert or by EMS or gamma-rays, in the 49F region mapped the latheo mutation to one vital complementation group. Flies heterozygous for latheoP1 and one of two EMS lethal alleles or one lethal excision allele also show the behavioral deficits, thereby demonstrating that the behavioral and lethal phenotypes co-map to the same locus.     

Developmental genetic analysis of lethal alleles at the ewg locus and their effects on muscle development in Drosophila melanogaster

The recessive X-linked mutation erect wing (ewg), in Drosophila melanogaster, was characterized as a flightless behavioral mutant which specifically lacked the dorsal longitudinal flight muscles [1]. This mutation was mapped distal to the X chromosomal locus yellow, and further to the cytological segment 1 A 1 to 1 B2-3 [2]. Several lethal complementation groups have been mapped to this interval [3]. Our complementation tests show that ewg is allelic to one lethal complementation group in the region 1 A 1 to 1 B2-3. A further analysis of ewg and several lethal alleles isolated at this locus was undertaken in the present investigation. Most of the lethal alleles at this locus lead to a late embryonic or early larval lethal phase, indicating that the ewg⁺ gene product is necessary for the development of more than just the dorsal longitudinal flight muscles. Intragenic complementation was observed for some of the ewg lethal alleles. Genetic mosaics with ewg lethal alleles showed that mutant cell clones in cuticular structures are viable. Mosaic analysis is consistent with a mesodermal defect associated with the locus.     

Mutations affecting skeletal muscle myofibril structure in the zebrafish

We describe embryonic lethal mutations in the zebrafish, Brachydanio rerio, which affect organization of skeletal muscle myofibrils. The mutations, fub-1(b45) and fub-1(b126), were independently isolated from progeny of gamma-irradiated females. Each segregates as a single recessive gene: b45 is located about 23 map units from its centromere. The b126 mutation has a similar but slightly larger apparent gene-centromere distance and a less severe phenotype. The two mutations fail to complement, suggesting that they are allelic. Homozygous b45 mutant embryos are paralyzed, and their axial skeletal muscle cells are unstriated, containing severely disorganized myofibrillar components. Gel-electrophoretic comparisons of b45 mutant and wild-type muscle proteins failed to reveal absent or altered major myofibrillar proteins. Embryos genetically mosaic for b45 were also phenotypically mosaic, suggesting that the defect is cell-autonomous. We suggest that these mutations identify a gene required for proper organization of skeletal muscle myofibrils, and that the more severe mutation may represent a null allele.     

Multiple Allele Approach to the Study of Genes in DROSOPHILA MELANOGASTER That Are Involved in Imaginal Disc Development

The phenotypes of five different lethal mutants of Drosophila melanogaster that have small imaginal discs were analyzed in detail. From these results, we inferred whether or not the observed imaginal disc phenotype resulted exclusively from a primary imaginal disc defect in each mutant. To examine the validity of these inferences, we employed a multiple-allele method. Lethal alleles of the five third-chromosome mutations were identified by screening EMS-treated chromosomes for those which fail to complement with a chromosome containing all five reference mutations. Twenty-four mutants were isolated from 13,197 treated chromosomes. Each of the 24 was then tested for complementation with each of the five reference mutants. There was no significant difference in the mutation frequencies at these five loci. The stage of lethality and the imaginal disc morphology of each mutant allele were compared to those of its reference allele in order to examine the range of defects to be found among lethal alleles of each locus. In addition, hybrids of the alleles were examined for intracistronic complementation. For two of the five loci, we detected no significant phenotypic variation among lethal alleles. We infer that each of the mutant alleles at these two loci cause expression of the null activity phenotype. However, for the three other loci, we did detect significant phenotypic variation among lethal alleles. In fact, one of the mutant alleles at each of these three loci causes no detectable imaginal disc defect. This demonstrates that attempting to assess the developmental role of a gene by studying a single mutant allele may lead to erroneous conclusions. As a byproduct of the mutagenesis procedure, we have isolated two dominant, cold-sensitive mutants.     

Interallelic Complementation among Der/Flb Alleles: Implications for the Mechanism of Signal Transduction by Receptor-Tyrosine Kinases

The large number of available embryonic lethal alleles in the Drosophila EGF receptor homolog (DER)/faint little ball locus allowed us to test the possibility of positive or negative interactions among different DER alleles. These interactions were monitored by examining the embryonic cuticular phenotypes of different heteroallelic combinations. Several positive interactions were identified, while negative interactions were restricted to a single allele. This is the first example of positive interactions within the same cell type among alleles of a receptor tyrosine kinase gene. The basis for these interactions is likely to arise from the mechanism of signal transduction by receptor tyrosine kinases, which involves receptor aggregation. A combination of two different DER mutant proteins defective in temporally distinct stages of the signal transduction process, may thus form a functional heterodimer. The mutation sites in four alleles showing positive interactions were localized. They identify regions within the protein which are likely to be important for these temporally distinct signal transduction processes.     

X-Linked Female-Sterile Loci in DROSOPHILA MELANOGASTER

We have examined the number of X-linked loci specifically required only during oogenesis. Complementation analyses among female-sterile (fs) mutations obtained in two mutagenesis screens--GANS' and MOHLER's--indicate that any fs locus represented by two or more mutant alleles in GANS' collection are usually present in MOHLER's collection. However, when a locus is represented by a single allele in one collection, it is generally not present in the other collection. We propose that this discrepancy is due to the fact that most "fs loci" represented by less than two mutant alleles are, in fact, vital (zygotic lethal) genes, and that the fs alleles are hypomorphic mutations of such genes. In support of this hypothesis we have identified lethal alleles at 12 of these "fs loci." The present analysis has possibly identified all maternal-effect lethal loci detectable by mutations on the X chromosome and has allowed us to reevaluate the number of "ovary-specific fs" loci in the Drosophila genome. Finally, germline clone analysis of a large number of fs mutations was performed in order to estimate the relative contribution of germline and somatic cell derivatives to oogenesis and to embryonic development. All the maternal-effect lethal loci tested are germline-dependent.     

Genetical analysis of visual system disorganizer (vid), a new gene involved in normal development of eye and optic lobe of the brain in Drosophila melanogaster

A neuroanatomical screening of a collection of P-element mutagenized flies has been carried out with the aim of finding new mutants affecting the optic lobe of the adult brain in Drosophila melanogaster. We have identified a new gene that is involved in the development of the adult axon array in the optic ganglia and in the ommatidia assembly. We have named this locus visual system disorganizer (vid). Reversional mutagenesis demonstrated that the vid mutant was the result of a P-element insertion in the Drosophila genome and allowed us to generate independent alleles, some of which resulted in semilethality, like the vid original mutant, while the others were completely lethal. A genetic somatic mosaic analysis indicated that the vid gene is required in the eye for its normal development by inductive effects. This analysis also suggests an inductive effect of the vid gene on the distal portion of the optic lobe, particularly the lamina and the first optic chiasma. Moreover, the absence of mutant phenotype in the proximal region of the optic ganglia, including the medulla, the second optic chiasma, and the lobula complex underlying mosaic eyes, is suggestive of an autonomously acting mechanism of the vid gene in the optic lobe. The complete or partial lethality generated by different mutations at the vid locus suggests that this gene's role may not be limited to the visual system, but may also affect a vital function during Drosophila development.     

Coordinately and Differentially Mutable Activities of Torpedo, the Drosophila Melanogaster Homolog of the Vertebrate Egf Receptor Gene

The torpedo (top) locus of Drosophila encodes the fruitfly homolog of the vertebrate epidermal growth factor receptor gene and the neu proto-oncogene. We have isolated 13 top alleles in a screen for mutations failing to complement the female sterility of top, a recessive maternal effect allele that disrupts the establishment of the dorsoventral pattern of the egg shell and embryo. Several alleles recovered in this screen are zygotic lethal mutations; genetic analysis of these alleles has demonstrated that top is allelic to the embryonic lethal locus faint little ball. The 13 mutations recovered in our screens and 19 previously isolated top alleles have been genetically characterized through complementation tests with a series of hypomorphic and amorphic alleles. Nearly every top allele fails to complement the maternal effect sterility of top. Complementation tests show that the gene is required not only for oogenesis and embryogenesis, but also for pupal viability, for the growth of certain imaginal discs and for the patterning of specific ectodermal derivatives of the imaginal discs. Complementation analysis further demonstrates that the top lesions can be divided into general phenotypic categories: alleles affecting all gene activities in a coordinate manner, alleles preferentially affecting embryogenesis, alleles preferentially retaining oogenesis activity and alleles differentially affecting the development of specific imaginal disc derivatives. Correlations observed between the various developmental defects produced by top lesions suggest that the gene possesses several differentially, though not independently, mutable activities.     

Mating type control in Saccharomyces cerevisiae: a frameshift mutation at the common DNA sequence, X, of the HML alpha locus.

A mutation defective in the homothallic switching of mating type alleles, designated hml alpha-2, has previously been characterized. The mutation occurred in a cell having the HO MATa HML alpha HMRa genotype, and the mutant culture consisted of ca. 10% a mating type cells, 90% nonmater cells of haploid cell size, and 0.1% sporogenous diploid cells. Genetic analyses revealed that nonmater haploid cells have a defect in the alpha 2 cistron at the MAT locus. This defect was probably caused by transposition of a cassette originating from the hml alpha-2 allele by the process of the homothallic mating type switch. That the MAT locus of the nonmater cells is occupied by a DNA fragment indistinguishable from the Y alpha sequence in electrophoretic mobility was demonstrated by Southern hybridization of the EcoRI-HindIII fragment encoding the MAT locus with a cloned HML alpha gene as the probe. The hml alpha-2 mutation was revealed to be a one-base-pair deletion at the ninth base pair in the X region from the X and Y boundary of the HML locus. This mutation gave rise to a shift in the open reading frame of the alpha 2 cistron. A molecular mechanism for the mating type switch associated with the occurrence of sporogenous diploid cells in the mutant culture is discussed.     

Induction of recessive lethal and specific locus mutations in the zebrafish with ethyl nitrosourea.

Recessive lethal mutations and mutations at the gol-1 locus were induced in the zebrafish by exposure of mature sperm to the alkylating agent ethyl nitrosourea (ENU). Embryonic lethal phenotypes were recognized among the parthenogenetic progeny of mutagenized animals or among the progeny of daughters of mutagenized animals. Novel specific locus mutations were identified by the failure of mutagenized chromosomes to complement pre-existing mutant alleles at the gol-1 locus. Each mutagenized individual harboured approximately 10 embryonic lethal mutations in its germ line and about 1 in 500 mutagenized animals harboured a new mutation at the gol-1 locus. Three lines of evidence indicate that the majority of mutations that were recovered following treatment of mature sperm with ENU were probably point mutations. First, the soma and germ lines of mutagenized animals were mosaic, as expected following simple alkylation of sperm DNA. Second, mutations induced by ENU at the gol-1 locus affected pigmentation but not viability, unlike the majority of mutations induced at this locus with gamma-irradiation. Third, the ratio of specific locus:recessive lethal mutations induced by ENU was approximately 50-fold lower than the ratio observed following mutagenesis with gamma-rays. Comparison of the incidence with which embryonic recessive lethal mutations were induced with the incidence with which specific locus mutations arose indicates that there are greater than 5000 genes essential to the development and viability of the zebrafish embryo.     

Dominance Modifiers in NEUROSPORA CRASSA: Phenocopy Selection and Influence on Certain Ascus Mutants

When homozygous in zygotes, mutant alleles at the peak locus in linkage group V of Neurospora crassa initiate aberrant asci that are nonlinear, in contrast to the linear asci characteristic of wild type. Most mutant alleles are recessive, inasmuch as crosses of the mutant strains with wild type give linear asci. However, five different mutant alleles, when heterozygous with the wild-type allele, act in varying degrees as zygote dominants, initiating both linear and nonlinear asci, the relative proportions depending on the allele. Five modifiers that act on the dominance relationships of at least one of the five possible heterozygotes of a dominant peak and its wild-type allele have been characterized, four of them having been obtained by selection directed against a phenocopy of these mutants induced by treatment of wild type with l-sorbose. The pattern of modifier specificity observed among the various dominant peak heterozygotes indicates that the phenotypic effects are produced by a complex relationship between the modifiers and the dominant peak alleles in relation to their wild-type allele. In all but two cases the direction of modification, where present, is towards decreasing the dominance of the mutant allele in the heterozygote, evidenced by an increase in the percentage of linear asci when compared with control data. The modifiers exert their maximum modification when they themselves are heterozygous with their wild-type alleles and when the dominant peak allele is heterozygous with its wild-type allele. No modification occurs when heterozygous modifiers are included in zygotes homozygous for a dominant peak allele, reinforcing the notion that the modifiers act on the dominance relationship existent between a dominant peak allele and its wild-type allele, rather than influencing some activity of the mutant allele itself. The modifiers have no detectable effect of their own on ascus morphology, since homozygous modifier zygotes initiate entirely linear asci when only wild-type alleles of peak are present in the zygotes. Their only detectable effect, other than dominance modification, appears to be in conferring sorbose resistance to the mycelium. The modifiers are unlinked to the peak locus, and, except for two of them, they are nonallelic.     

Genetic study of a lethal necrosis to soybean mosaic virus in PI 507389 soybean

PI 507389 soybean [Glycine max (L.) Merr.], a large-seeded line from Japan, exhibits a rapid, lethal, necrotic response to strains G1, G2, G5, and G6 of soybean mosaic virus (SMV). Unlike the hypersensitive necrotic reaction, this stem-tip necrosis can be a serious threat to soybean production. To investigate the genetic basis of lethal necrosis (LN), PI 507389 was crossed with the susceptible (S) cv. Lee 68 and with resistant (R) lines PI 96983, cv. York, and cv. Marshall, which carry single dominant genes for SMV resistance at the Rsv1 locus. F(1) plants, F(2) populations, and F(2:3) lines were inoculated with G1 and G6 in the greenhouse or in the field. Results indicated that LN is controlled by a single gene allelic to Rsv1, and this allele in PI 507389 is recessive to R alleles in PI 96983, York, and Marshall. The LN allele is codominant with the allele for S, for the heterozygotes showed a mixed phenotype of both necrosis (N) and mosaic (M) symptoms (NM). The LN allele becomes recessive to the S allele as the mixed NM shifts to S at a later stage in response to more virulent strains. The gene symbol Rsv1-n is assigned for the allele conferring LN in PI 507389. Rsv1-n is the only allele at the Rsv1 locus conditioning N to G1 and no R to any other SMV strains, and thus a unique genotype for SMV strain differentiation. The phenotypic expression of heterozygotes and the dominance relationships among R, N, and S depend on the virulence of SMV strains, source of alleles, and developmental stage.     

MASTR: a technique for mosaic mutant analysis with spatial and temporal control of recombination using conditional floxed alleles in mice

Mosaic mutant analysis, the study of cellular defects in scattered mutant cells in a wild-type environment, is a powerful approach for identifying critical functions of genes and has been applied extensively to invertebrate model organisms. A highly versatile technique has been developed in mouse: MASTR (mosaic mutant analysis with spatial and temporal control of recombination), which utilizes the increasing number of floxed alleles and simultaneously combines conditional gene mutagenesis and cell marking for fate analysis. A targeted allele (R26(MASTR)) was engineered; the allele expresses a GFPcre fusion protein following FLP-mediated recombination, which serves the dual function of deleting floxed alleles and marking mutant cells with GFP. Within 24 hr of tamoxifen administration to R26(MASTR) mice carrying an inducible FlpoER transgene and a floxed allele, nearly all GFP-expressing cells have a mutant allele. The fate of single cells lacking FGF8 or SHH signaling in the developing hindbrain was analyzed using MASTR, and it was revealed that there is only a short time window when neural progenitors require FGFR1 for viability and that granule cell precursors differentiate rapidly when SMO is lost. MASTR is a powerful tool that provides cell-type-specific (spatial) and temporal marking of mosaic mutant cells and is broadly applicable to developmental, cancer, and adult stem cell studies.     

A role for N-cadherin in mesodermal morphogenesis during gastrulation

Cell adhesion molecules mediate numerous developmental processes necessary for the segregation and organization of tissues. Here we show that the zebrafish biber (bib) mutant encodes a dominant allele at the N-cadherin locus. When knocked down with antisense oligonucleotides, bib mutants phenocopy parachute (pac) null alleles, demonstrating that bib is a gain-of-function mutation. The mutant phenotype disrupts normal cell-cell contacts throughout the mesoderm as well as the ectoderm. During gastrulation stages, cells of the mesodermal germ layer converge slowly; during segmentation stages, the borders between paraxial and axial tissues are irregular and somite borders do not form; later, myotomes are fused. During neurulation, the neural tube is disorganized. Although weaker, all traits present in bib mutants were found in pac mutants. When the distribution of N-cadherin mRNA was analyzed to distinguish mesodermal from neuroectodermal expression, we found that N-cadherin is strongly expressed in the yolk cell and hypoblast in the early gastrula, just preceding the appearance of the bib mesodermal defects. Only later is N-cadherin expressed in the anlage of the CNS, where it is found as a radial gradient in the forming neural plate. Hence, besides a well-established role in neural and somite morphogenesis, N-cadherin is essential for morphogenesis of the mesodermal germ layer during gastrulation.     

The cfy mutation disrupts cell divisions in a stage-dependent manner in zebrafish embryos

The zebrafish curly fry (cfy) mutation leads to embryonic lethality and abnormal cell divisions starting at 12-14 h postfertilization (hpf) during neural tube formation. The mitotic defect is seen in a variety of tissues including the central nervous system (CNS). In homozygous mutant embryos, mitoses are disorganized with an increase in mitotic figures throughout the developing neural tube. One consequence of aberrant mitoses in cfy embryos is an increase in cell death. Despite this, patterning of the early CNS is relatively unperturbed with distribution of the early, primary neurons indistinguishable from that of wild-type embryos. At later stages, however, the number of neurons was dramatically decreased throughout the CNS. The effect on neurons in older cfy embryos but not young ones correlates with the time of birth of neurons: primary neurons are born before the action of the cfy gene and later neurons after. Presumably, death of neuronal progenitors that divide beginning at the neural keel stage or death of their neuronal progeny accounts for the diminution of neurons in older mutant embryos. In addition, oligodendrocytes, which also develop late in the CNS, are greatly reduced in number in cfy embryos due to an apparent decrease in oligodendrocyte precursors. Genetic mosaic analysis demonstrates that the mutant phenotype is cell-autonomous. Furthermore, there are no obvious defects in apical/basal polarity within the neuroepithelium, suggesting that the cfy gene is not critical for epithelial polarity and that polarity defects are unlikely to account for the increased mitotic figures in mutants. These results suggest that the cfy gene regulates mitosis perhaps in a stage-dependent manner in vertebrate embryos.