Embryonic Lethal Mutations Research Articles

Hedgehog-Gli Signaling Pathway Inhibitors as Anticancer Agents

Cancer drug discovery has undergone a paradigm change over the past few years, from predominantly cytotoxic agent-based therapy to therapy aimed at genetic and molecular targets, thanks to a growing understanding of the genes and pathways responsible for cancer initiation and progression and to new drug discovery technologies. The success of drugs like trastuzumab, imatinib, gefitinib, and erlotinib has demonstrated that the targeting of specific oncogenic signal transduction pathways can be clinically useful.1 One such pathway, the Hedgehog-Glioma-associated oncogene homolog zinc finger protein (Hh-Gli)a signaling pathway, has attracted drug discovery scientists for the past decade. Hh-Gli signaling plays an important role in the embryonic patterning and development of many tissues and somatic structures as well as maintaining and repairing mature tissues in adults.2-4 Uncontrolled activation of the Hh-Gli pathway has been implicated in several cancers, including medulloblastoma, rhabdomyosarcoma, melanoma, basal cell carcinoma, and breast, lung, liver, stomach, prostate, and pancreatic cancers. 2, 5-8 Inhibition of the aberrant Hh-Gli pathway (Figure 1) has thus emerged as an attractive target for anticancer therapy.9-11 One Hh pathway inhibitor has shown promising results in phase I clinical trials and is proceeding to phase II12 studies, and two other compounds have entered phase I clinical trials.13, 14 In this article, we review the medicinal chemistry efforts to identify and design inhibitors of Hh-Gli signaling and present a perspective of future developments in this dynamic field. We also present a brief overview of the role of Hh-Gli signaling pathway in normal development and cancer. Figure 1 Hedgehog pathway activators and inhibitors The hedgehog (Hh) gene was first identified during a search for embryonic lethal mutants of Drosophila melanogaster, which found that mutation of Hh resulted in altered segment patterning of the larva.15 Subsequently the gene was identified in many other invertebrates and vertebrates, including humans. Three mammalian counterparts of the Hh gene, termed Sonic hedgehog (Shh), Dessert hedgehog (Dhh), and Indian hedgehog (Ihh), were identified by combined screening of mouse genomic and cDNA libraries.16 Hh undergoes multiple processing events, including autocatalytic cleavage of the C-terminal domain combined with addition of a cholesterol moiety at the cleavage site, and an N-terminal palmitoylation, to generate the active ligand.17-19 The receptor of secreted Hh protein is the multipass transmembrane protein Patched (Ptch). Of the two vertebrate homologs of Ptch, Ptch1 and Ptch2, the role of Ptch1 is better understood. In the absence of Hh ligand, Ptch inhibits the activity of the downstream effector Smoothened (Smo). The binding of Hh inactivates Ptch, resulting in activation of Smo.20 In Drosophila, a complex of proteins comprising Fused (Fu), Suppressor of Fused (SuFu), and Costal-2 (Cos2) mediates signaling downstream of Smo and is aided by several kinases, such as protein kinase A (PKA), glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1). Mammalian homologs of Fu and Cos2 have not yet been identified, suggesting that the signaling mechanisms differ in mammals and Drosophila. 21, 22 Several mammalian-specific kinases that are required for Shh signaling have been identified.23-25 These proteins modulate the function of Gli (Ci in Drosophila), the only transcription factor identified to date that operates directly downstream of Hh. The first vertebrate Gli gene to be discovered was human Gli1, which was amplified about 50-fold in a malignant glioma.26 Vertebrates have three Gli proteins (Gli1, Gli2 and Gli3), all of which have five highly conserved tandem zinc fingers, a fairly conserved N-terminal domain, several potential PKA sites, and a number of additional small conserved regions in the C-terminal end. Despite these similarities, the functions of the Gli subtypes differ. Both Gli2 and Gli3 contain activation and repressor domains. Consequently, in the absence of upstream Hh signal, full-length Gli3 and, to a lesser extent, Gli2, are constitutively cleaved to generate a truncated repressor form.27-29 Hh signaling inhibits this cleavage, resulting in full-length Gli2 and Gli3, which have activator function. Gli1, in contrast, does not undergo proteolytic cleavage and acts as a constitutive activator.27 The transcription of Gli1 gene is initiated by Hh and is also controlled by Gli3.27 Target genes of the Hh pathway other than Gli1 include Ptch, several Wnt and TGFβ superfamily proteins, cell cycle proteins such as cyclin D, and stem-cell marker genes such as NANOG and SOX2.30, 31 Investigators are now attempting to comprehensively identify the Gli1-target genes.32, 33

A Novel Transcription Factor, zbtb11 Is Critical for Neutrophil Development in Zebrafish

Zinc finger and BTB domain containing proteins (BTB-ZF) are transcriptional repressors from a family including members with critical roles in hematopoiesis and oncogenesis (BCL6, PLZF, HIC). In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen for defects in myeloid development, we identified a neutropenic zebrafish mutant with a mutation in zbtb11, a novel BTB-ZF transcription factor, suggesting that zbtb11 is critical for normal neutrophil development.The mutant was found in a gynogenetic haploid ENU screen for defective expression of genes along the developmental pathway from mesoderm to mature neutrophil (deltaC, spi1 and myeloperoxidase/mpx), undertaken to search for novel genetic regulators of myelopoiesis in an unbiased fashion. Since zebrafish are poikilothermic, embryos were screened at 33°C to maximise recovery of temperature dependant alleles; marsanne (man) was one mutant pedigree recovered. man is an early embryonic lethal mutant with normal scl and spi1 expression but markedly reduced numbers of mpx expressing cells. Erythropoiesis is unaffected. Reduced numbers of Sudan Black stained cells demonstrate that man is deficient in neutrophils themselves, not just mpx transcripts. man mutants also develop brain and spinal cord degeneration with hydrocephalus due to apoptosis. man is a temperature sensitive allele i.e. numbers of mpx expressing cells per embryo decrease at higher (restrictive) temperatures and are partially restored at lower (permissive) temperatures:Water temperatureNumber of mpx expressing cells/embryo (mean ± SD, 48hpf)man/man+/?22°C75±1589±1428°C42±1081±1233°C27±1181±22Hence man serves as a conditional allele, allowing manipulation of protein function via manipulation of water temperature. man was mapped to a 40kb region on chromosome 6 containing a single gene, zbtb11. Sequencing of man and WT zbtb11 cDNA revealed a missense mutation (346T->A, 116Cys->Ser) affecting a cysteine residue conserved throughout vertebrate evolution. Pedigree analysis proved this substitution was introduced by ENU mutagenesis and segregated absolutely with the man phenotype in 3863 mutant embryos. Genetic tests implicated this zbtb11 mutation as causing the man phenotype, zbtb11 knockdown with an antisense morpholino oligonucleotide recapitulated the mpx deficiency and other features of the man phenotype. Injection of WT but not mutant zbtb11 mRNA into man embryos completely rescued mpx expression (125 +/−38 mpx positive cells per embryo vs. 67+/−35, p<0.001; uninjected WT 109 +/19, mean +/− SD, 48hpf) and all other aspects of the man phenotype. Since the mutant Zbtb11 protein is inactive in this assay, we hypothesise the amino acid substitution in the N-terminus of the protein compromises function. In view of the temperature sensitive nature of man, this may result from an effect on weak bonding, differentially affecting protein structure at permissive vs. restrictive temperatures. Zebrafish zbtb11 encodes a 1146 amino acid protein with a predicted N-terminal BTB (also known as POZ) domain and 12 C-terminal C2H2 (Kruppel-type) zinc fingers. The BTB domain is a highly conserved, approximately 100 amino acid domain, mediating protein-protein interaction and dimerization. The zebrafish protein is approximately 75% identical to mouse and human ZBTB11 across its BTB domain and zinc fingers. ZBTB11 is expressed physiologically in human hematopoietic cells (Ferrari F et al. BMC Genomics. 2007) and mouse embryonic brain (Gray PA et al. Science. 2004). It appears to be over-expressed in K562 CML cells compared to other acute leukemia and lymphoma lines (Su AI et al. PNAS 2002), suggesting a possible role in oncogenesis. The function of mammalian ZBTB11 is unknown and there are no mammalian models of ZBTB11 dysfunction. The rescue of man by WT zbtb11 forms the basis of a bioassay to test modified Zbtb11 proteins, and a whole animal assay for chemical modifiers of Zbtb11 function. Rescue of man with human ZBTB11 is being attempted to assess functional conservation and to provide a heterologous mammalian bioassay. Temperature-shift experiments exploiting the temperature sensitivity of man should define the time-dependant requirement for Zbtb11 during development. man provides a unique reagent with which to advance understanding of Zbtb11 biochemistry and biology in vivo.

Primers on Molecular Pathways GLI: More than Just Hedgehog?

GLI proteins, highly conserved in vertebrates and invertebrates, are critical in modulating embryonic development and adult tissue homeostasis. These proteins are zinc-finger-containing transcription factors that were originally identified by genetic screening of embryonic lethal mutants of Drosophila melanogaster. Alterations in GLI activity can lead to tumor development in tissues of different origin including the pancreas. GLI activity is mainly regulated by the Hedgehog pathway, via a ligand-receptor complex that triggers a signaling cascade that activates GLI transcription factors, which in turn regulate gene expression, an essential step of Hegdehog-mediated cellular effects. Interestingly, recent reports show the ability of other signaling cascades to modulate GLI function in cancer cells including RAS and TGF-β, two pathways implicated in pancreatic carcinogenesis.Thus, these findings suggest that GLI proteins are not an exclusive downstream target of Hedgehog but rather a common effector of a network of signaling pathways controlling pancreatic Carcinogenesis.

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

The BRCT-Domain Containing Protein PTIP Links PAX2 to a Histone H3, Lysine 4 Methyltransferase Complex

The MLL family of histone methyltransferases maintains active chromatin domains by methylating histone H3 on lysine 4 (H3K4). How MLL complexes recognize specific chromatin domains in a temporal and tissue-specific manner remains unclear. We show that the DNA-binding protein PAX2 promotes assembly of an H3K4 methyltransferase complex through the ubiquitously expressed nuclear factor PTIP (pax transcription activation domain interacting protein). PTIP copurifies with ALR, MLL3, and other components of a histone methyltransferase complex. PTIP promotes assembly of the ALR complex and H3K4 methylation at a PAX2-binding DNA element. Without PTIP, Pax2 binds to this element but does not assemble the ALR complex. Embryonic lethal ptip-null mutants and conditional mutants both show reduced levels of methylated H3K4. Thus, PTIP bridges DNA-binding developmental regulators to histone methyltransferase-dependent epigenetic regulation.

The emergence of patterned movement during late embryogenesis of Drosophila

Larval behavioral patterns arise in a gradual fashion during late embryogenesis as the innervation of the somatic musculature and connectivity within the central nervous system develops. In this paper, we describe in a quantitative manner the maturation of behavioral patterns. Early movements are locally restricted "twitches" of the body wall, involving single segments or parts of segments. These twitches occur at a low frequency and have low amplitude, reflecting weak muscle contractions. Towards later stages twitches increase in frequency and amplitude and become integrated into coordinated movements of multiple segments. Most noticeable among these is the peristaltic wave of longitudinal segmental contractions by which the larva moves forward or backward. Besides becoming more complex as development proceeds, embryonic movements also acquire a pronounced rhythm. Thus, late embryonic movements occur in bursts, with phases of frequent movement separated by phases of no movement at all; early movements show no such periodicity. These data will serve as a baseline for future studies that address the function of embryonic lethal genes controlling neuronal connectivity and larval behavior. We have analyzed behavioral abnormalities in two embryonic lethal mutations with severe neural defects, tailless (tll), which lacks the protocerebrum, and glial cells missing (gcm), in which glial cells are absent. Our results reveal prominent alterations in embryonic motility for both of these mutations, indicating that the protocerebrum and glial cells play a crucial role in the neural mechanism controlling larval movement in Drosophila.

The role of the SPT6 chromatin remodeling factor in zebrafish embryogenesis

Somitogenesis is a highly controlled process that results in segmentation of the paraxial mesoderm. Notch pathway activity in the presomitic mesoderm is fundamental for management of synchronized gene expression which is necessary for regulation of somitogenesis. We have isolated an embryonic lethal mutation, SBU2, that causes somite formation defects very similar to Notch pathway mutants. SBU2 mutants generate only 6–7 asymmetrically arranged somites. However, in contrast to Notch pathway mutants, these mutants do not maintain previously formed somite boundaries and by 24 hpf, almost no somite boundaries remain. Other developmental processes disrupted in SBU2 mutants include tail morphogenesis, muscle fiber elongation, pigmentation, circulatory system development and neural differentiation. We demonstrated that these defects are the result of a nonsense mutation within the spt6 gene. spt6 encodes a transcription elongation factor that genetically interacts with the Paf-1 chromatin remodeling complex. SBU2 mutant phenotypes could be rescued by microinjection of spt6 mRNA and microinjection of spt6 morpholinos phenocopied the mutation. Our real-time PCR analysis revealed that Spt6 is essential for the transcriptional response to activation of the Notch pathway. Analysis of sbu2;mib double mutants indicates that Spt6 deficiency suppresses the neurogenic effects of the mib. Altogether, these results demonstrate that Spt6 is critical for somite formation in zebrafish and suggest that some defects observed in spt6 mutants result from alterations in Notch signaling. However, additional Spt6 mutant phenotypes are likely caused by vital functions of Spt6 in other pathways.

The modifier ofMin2 (Mom2) locus: Embryonic lethality of a mutation in theAtp5a1gene suggests a novel mechanism of polyp suppression

Inactivation of the APC gene is considered the initiating event in human colorectal cancer. Modifier genes that influence the penetrance of mutations in tumor-suppressor genes hold great potential for preventing the development of cancer. The mechanism by which modifier genes alter adenoma incidence can be readily studied in mice that inherit mutations in the Apc gene. We identified a new modifier locus of ApcMin-induced intestinal tumorigenesis called Modifier of Min 2 (Mom2). The polyp-resistant Mom2R phenotype resulted from a spontaneous mutation and linkage analysis localized Mom2 to distal chromosome 18. To obtain recombinant chromosomes for use in refining the Mom2 interval, we generated congenic DBA.B6 ApcMin/+, Mom2R/+ mice. An intercross revealed that Mom2R encodes a recessive embryonic lethal mutation. We devised an exclusion strategy for mapping the Mom2 locus using embryonic lethality as a method of selection. Expression and sequence analyses of candidate genes identified a duplication of four nucleotides within exon 3 of the alpha subunit of the ATP synthase (Atp5a1) gene. Tumor analyses revealed a novel mechanism of polyp suppression by Mom2R in Min mice. Furthermore, we show that more adenomas progress to carcinomas in Min mice that carry the Mom2R mutation. The absence of loss of heterozygosity (LOH) at the Apc locus, combined with the tendency of adenomas to progress to carcinomas, indicates that the sequence of events leading to tumors in ApcMin/+ Mom2R/+ mice is consistent with the features of human tumor initiation and progression.

L2dtl Is Essential for Cell Survival and Nuclear Division in Early Mouse Embryonic Development

l(2)dtl (lethal (2) denticleless), is an embryonic lethal homozygous mutation initially identified in Drosophila melanogaster that produces embryos that lack ventral denticle belts. In addition to nucleotide sequence, bioinformatic analysis has revealed a conservation of critical functional motifs among the human L2DTL, mouse L2dtl, and Drosophila l(2)dtl proteins. The function of the L2DTL protein in the development of mammalian embryos was studied using targeted disruption of the L2dtl gene in mice. The knock-out resulted in early embryonic lethality. L2dtl-/- embryos were deformed and terminated development at the 4-8-cell stage. Microinjection of a small interfering RNA (siRNA) vector (siRNA-L2dtl) into the two-cell stage nuclei of wild-type mouse embryos led to cell cycle progression failure, termination of cell division, and, eventually, embryonic death during the preimplantation stage. Morphological studies of the embryos 54 h after injection showed fragmentation of mitotic chromosomes and chromosomal lagging, hallmarks of mitotic catastrophe. The siRNA-L2dtl-treated embryos eventually lysed and failed to develop into blastocysts after 72 h of in vitro culturing. However, the embryos developed normally after they were microinjected into one nucleus of the two-celled embryos. The siRNA studies in HeLa cells showed that L2dtl protein depletion results in multinucleation and down-regulation of phosphatidylinositol 3-kinase, proliferating cell nuclear antigen, and PTTG1/securin, which might partially explain the mitotic catastrophe observed in L2dtl-depleted mouse embryos. Based on these findings, we conclude that L2dtl gene expression is essential for very early mouse embryonic development.

Silencing by plant Polycomb-group genes requires dispersed trimethylation of histone H3 at lysine 27

The plant Polycomb-group (Pc-G) protein CURLY LEAF (CLF) is required to repress targets such as AGAMOUS (AG) and SHOOTMERISTEMLESS (STM). Using chromatin immunoprecipitation, we identify AG and STM as direct targets for CLF and show that they carry a characteristic epigenetic signature of dispersed histone H3 lysine 27 trimethylation (H3K27me3) and localised H3K27me2 methylation. H3K27 methylation is present throughout leaf development and consistent with this, CLF is required persistently to silence AG. However, CLF is not itself an epigenetic mark as it is lost during mitosis. We suggest a model in which Pc-G proteins are recruited to localised regions of targets and then mediate dispersed H3K27me3. Analysis of transgenes carrying AG regulatory sequences confirms that H3K27me3 can spread to novel sequences in a CLF-dependent manner and further shows that H3K27me3 methylation is not sufficient for silencing of targets. We suggest that the spread of H3K27me3 contributes to the mitotic heritability of Pc-G silencing, and that the loss of silencing caused by transposon insertions at plant Pc-G targets reflects impaired spreading.

Hypomorphic Mutation of the TALE Gene Prep1 (pKnox1) Causes a Major Reduction of Pbx and Meis Proteins and a Pleiotropic Embryonic Phenotype

The interaction of Prep1 and Pbx homeodomain transcription factors regulates their activity, nuclear localization, and likely, function in development. To understand the in vivo role of Prep1, we have analyzed an embryonic lethal hypomorphic mutant mouse (Prep1(i/i)). Prep1(i/i) embryos die at embryonic day 17.5 (E17.5) to birth with an overall organ hypoplasia, severe anemia, impaired angiogenesis, and eye anomalies, particularly in the lens and retina. The anemia correlates with delayed differentiation of erythroid progenitors and may be, at least in part, responsible for intrauterine death. At E14.5, Prep1 is present in fetal liver (FL) cMyb-positive cells, whose deficiency causes a marked hematopoietic phenotype. Prep1 is also localized to FL endothelial progenitors, consistent with the observed angiogenic phenotype. Likewise, at the same gestational day, Prep1 is present in the eye cells that bear Pax6, implicated in eye development. The levels of cMyb and Pax6 in FL and in the retina, respectively, are significantly decreased in Prep1(i/i) embryos, consistent with the hematopoietic and eye phenotypes. Concomitantly, Prep1 deficiency results in the overall decrease of protein levels of its related family member Meis1 and its partners Pbx1 and Pbx2. As both Prep1 and Meis interact with Pbx, the overall Prep1/Meis-Pbx DNA-binding activity is strongly reduced in whole Prep1(i/i) embryos and their organs. Our data indicate that Prep1 is an essential gene that acts upstream of and within a Pbx-Meis network that regulates multiple aspects of embryonic development.

Maintenance of segment and appendage primordia by the Tribolium gene knödel

For homeotic and segment-polarity genes in Drosophila, a switch in gene regulation has been described that distinguishes patterning and maintenance phases. Maintenance of segment and organ primordia involves secondary patterning and differentiation steps, as well as survival factors regulating proliferation and organ size. In a screen for embryonic lethal mutations in the flour beetle Tribolium castaneum, we have recovered two alleles of the knödel gene, which result in short, bag-like embryos. These embryos have severely reduced appendages and differentiate a cuticle that lacks most overt signs of segmentation. In addition, they lack bristles and display defects in the nervous system. Early patterning in knödel mutant embryos is normal up to the extended germ band stage, as indicated by the formation of regular even-skipped ( Tc'eve) and wingless ( Tc'wg) stripes. Afterwards, however, these patterns degenerate. Similarly, proximo-distal growth and patterning of limbs are nearly normal initially, but limb primordia shrink, and proximo-distal patterns degenerate, during subsequent stages. knödel could be a segment polarity gene required for segment border maintenance in both trunk and appendages. Alternatively, it may have a more general role in tissue or organ maintenance.

Random mutagenesis of proximal mouse chromosome 5 uncovers predominantly embryonic lethal mutations

A region-specific ENU mutagenesis screen was conducted to elucidate the functional content of proximal mouse Chr 5. We used the visibly marked, recessive, lethal inversion Rump White (Rw) as a balancer in a three-generation breeding scheme to identify recessive mutations within the approximately 50 megabases spanned by Rw. A total of 1003 pedigrees were produced, representing the largest inversion screen performed in mice. Test-class animals, homozygous for the ENU-mutagenized proximal Chr 5 and visibly distinguishable from nonhomozygous littermates, were screened for fertility, hearing, vestibular function, DNA repair, behavior, and dysmorphology. Lethals were identifiable by failure to derive test-class animals within a pedigree. Embryonic lethal mutations (total of 34) were overwhelmingly the largest class of mutants recovered. We characterized them with respect to the time of embryonic death, revealing that most act at midgestation (8.5-10.5) or sooner. To position the mutations within the Rw region and to guide allelism tests, we performed complementation analyses with a set of new and existing chromosomal deletions, as well as standard recombinational mapping on a subset of the mutations. By pooling the data from this and other region-specific mutagenesis projects, we calculate that the mouse genome contains approximately 3479-4825 embryonic lethal genes, or about 13.7%-19% of all genes.

Repression of Cell-Cell Fusion by Components of the C. elegans Vacuolar ATPase Complex

Cell-cell fusion initiates fertilization, sculpts tissues during animal development, reprograms stem cells to new differentiated states, and may be a key step in cancer progression. While cell fusion is tightly regulated, the mechanisms that limit fusion to appropriate partners are unknown. Here, we report that the fus-1 gene is essential to repress fusion of epidermal cells in C. elegans: in severe fus-1 mutants, all epidermal cells, except the lateral seam cells, inappropriately fuse into a single large syncytium. This hyperfusion requires EFF-1, an integral membrane protein essential for fusion of epidermal cells into discrete syncytia. FUS-1 is localized to the apical plasma membrane in all epidermal cells potentiated to undergo fusion, whereas it is virtually undetectable in nonfusing seam cells. fus-1 encodes the e subunit of the vacuolar H(+)-ATPase (V-ATPase), and loss of other V-ATPase subunits also causes widespread hyperfusion. These findings raise the possibility of manipulating cell fusion by altering V-ATPase activity.

Hypomorphic mutation in hnRNP U results in post-implantation lethality

The present study characterized an embryonic lethal mutation induced by insertion of the U3Neo gene trap retrovirus into an intron of the gene encoding heterogeneous ribonuclear protein U (Hnrnpu), which maps to the distal arm of mouse chromosome 1. Murine hnRNP U was found to be identical to the human protein at all but one of 341 amino acid residues. Embryos homozygous for the provirus showed obvious abnormalities after 6.5 days of development (E6.5) and were resorbed by E10.5. Expression of the inserted neomycin-resistance gene involved alternative splicing to a cryptic 3' splice site located in the neomycin resistance gene resulting in a hypomorphic mutation. Homozygous mutant cell lines isolated from preimplantation blastocysts expressed hnRNP U transcripts at levels 2 to 5 times lower than wild-type cells, suggesting that nearly wild-type levels of hnRNP U are required for embryonic development.

Functional analysis of seven genes encoding eight translation initiation factor 4E (eIF4E) isoforms in Drosophila

The Drosophila genome-sequencing project has revealed a total of seven genes encoding eight eukaryotic initiation factor 4E (eIF4E) isoforms. Four of them ( eIF4E-1,2, eIF4E-3, eIF4E-4 and eIF4E-5) share exon/intron structure in their carboxy-terminal part and form a cluster in the genome. All eIF4E isoforms bind to the cap (m 7GpppN) structure. All of them, except eIF4E-6 and eIF4E-8 were able to interact with Drosophila eIF4G or eIF4E-binding protein (4E-BP). eIF4E-1, eIF4E-2, eIF4E-3, eIF4E-4 and eIF4E-7 rescued a yeast eIF4E-deficient mutant in vivo. Only eIF4E-1 mRNAs and, at a significantly lower level, eIF4E3 and eIF4E-8 are expressed in embryos and throughout the life cycle of the fly. The transcripts of the remaining isoforms were detected from the third instar larvae onwards. This indicates the cap-binding activity relies mostly on eIF4E-1 during embryogenesis. This agrees with the proteomic analysis of the eIF4F complex purified from embryos and with the rescue of l(3)67Af, an embryonic lethal mutant for the eIF4E-1,2 gene, by transgenic expression of eIF4E-1. Overexpression of eIF4E-1 in wild-type embryos and eye imaginal discs results in phenotypic defects in a dose-dependent manner.

ClickersA Teaching Gimmick that Works

ClickersA Teaching Gimmick that Works

A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

Histological Observation of Eggs of the Embryonic Lethal Mutation Set in Bombyx mori

Histological Observation of Eggs of the Embryonic Lethal Mutation Set in Bombyx mori

Many ribosomal protein genes are cancer genes in zebrafish.

We have generated several hundred lines of zebrafish (Danio rerio), each heterozygous for a recessive embryonic lethal mutation. Since many tumor suppressor genes are recessive lethals, we screened our colony for lines that display early mortality and/or gross evidence of tumors. We identified 12 lines with elevated cancer incidence. Fish from these lines develop malignant peripheral nerve sheath tumors, and in some cases also other tumor types, with moderate to very high frequencies. Surprisingly, 11 of the 12 lines were each heterozygous for a mutation in a different ribosomal protein (RP) gene, while one line was heterozygous for a mutation in a zebrafish paralog of the human and mouse tumor suppressor gene, neurofibromatosis type 2. Our findings suggest that many RP genes may act as haploinsufficient tumor suppressors in fish. Many RP genes might also be cancer genes in humans, where their role in tumorigenesis could easily have escaped detection up to now.