Embryonic Lethal Mutations Research Articles

Gastrulation initiation in Caenorhabditis elegans requires the function of gad-1, which encodes a protein with WD repeats

Gastrulation in Caenorhabditis elegans is normally initiated by inward migration of the two gut precursor (E) cells at the 26-cell stage. A strong loss-of-function, temperature-sensitive, embryonic lethal mutation in the maternally required gene gad-1 ( gastrulation defective) prevents gastrulation initiation. In embryos from homozygous mutant gad-1 (ct226) hermaphrodites reared at 25°C, the E cellsdivide early with abnormal spindle orientations and fail to migrate into the embryo, and no subsequent gastrulation movements occur. These embryos continue to develop and differentiate the major cell types, but they undergo little morphogenesis. The temperature-sensitive period of the mutant is during early embryogenesis, prior to gastrulation onset. The predicted translation product of the cloned gad-1 gene includes six β-transducin-related repeats of the WD motif, which has been implicated in protein-protein interactions. The ct226 mutation alters a conserved residue in one of these repeats. Injection of gad-1 antisense RNA into wild-type hermaphrodites mimics the mutant phenotype in progeny embryos. We conclude that the gad-1 gene product is required for initiation of gastrulation in C. elegans.

An Arabidopsis embryonic lethal mutant with reduced expression of alanyl-tRNA synthetase gene.

In present paper, one of the T-DNA insertional embryonic lethal mutant of Arabidopsis is identified and designated as acd mutant. The embryo development of this mutant is arrested in globular stage. The cell division pattern is abnormal during early embryogenesis and results in disturbed cellular differentiation. Most of mutant embryos are finally degenerated and aborted in globular stage. However, a few of them still can germinate in agar plate and produce seedlings with shorter hypocotyl and distorted shoot meristem. To understand the molecular basis of the phenotype of this mutant, the joint fragment of T-DNA/plant DNA is isolated by plasmid rescue and Dig-labeled as probe for cDNA library screening. According to the sequence analysis and similarity searching, a 936 bp cDNA sequence (EMBL accession #: Y12555) from selected positive clone shows a 99.8% (923/925bp) sequence homology with Alanyl-tRNA Synthetase (AlaRS) gene of Arabidopsis thaliana. Furthermore, the data of in situ hybridization experiment indicate that the expression of AlaRS gene is weak in early embryogenesis and declines along with globular embryo 'development' in this mutant. Accordingly, the reduced expression of AlaRS gene may be closely related to the morphological changes in early embryogenesis of this lethal mutant.

Mutant Rescue by BAC Clone Injection in Zebrafish

Genes essential for vertebrate body plan specification, organ development, and organ function are likely to be shared between mammals and zebrafish, but only in zebrafish have large-scale, genome-wide mutagenesis screens been conducted to isolate embryonic lethal mutations. Discovering the roles played by these disrupted genes requires their molecular characterization, which would be facilitated by assaying large cloned genomic DNAs for their potential to rescue mutant phenotypes. Here we demonstrate that bacterial artificial chromosomes can rescue the phenotype offloating head(flh) mutants. Homozygousflhembryos lack a differentiated notochord and have a reduced, discontinuous floor plate. Mutant embryos injected with genomic clones containing theflh+gene often had stretches of several to many notochord cells overlaid by a row of floor-plate cells. In contrast, control mutant embryos injected with artificial chromosomes lacking theflh+gene failed to form notochord. We conclude that the injection of large-insert genomic clones will speed the isolation of zebrafish genes disrupted by mutation and hence the identification of gene functions necessary for development of vertebrate embryos.

Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development.

We disrupted the fibroblast growth factor (FGF) receptor 2 (FGFR2) gene by introducing a neo cassette into the IIIc ligand binding exon and by deleting a genomic DNA fragment encoding its transmembrane domain and part of its kinase I domain. A recessive embryonic lethal mutation was obtained. Preimplantation development was normal until the blastocyst stage. Homozygous mutant embryos died a few hours after implantation at a random position in the uterine crypt, with collapsed yolk cavity. Mutant blastocysts hatched, adhered, and formed a layer of trophoblast giant cells in vitro, but after prolonged culture, the growth of the inner cell mass stopped, no visceral endoderm formed, and finally the egg cylinder disintegrated. It follows that FGFR2 is required for early postimplantation development between implantation and the formation of the egg cylinder. We suggest that FGFR2 contributes to the outgrowth, differentiation, and maintenance of the inner cell mass and raise the possibility that this activity is mediated by FGF4 signals transmitted by FGFR2. The role of early FGF signaling in pregastrulation development as a possible adaptation to mammalian (amniote) embryogenesis is discussed.

3 Maternal Control of Pattern Formation in Early Caenorhabditis elegans Embryos

Genetic screens for recessive, maternal-effect, embryonic-lethal mutations have identified about 25 genes that control early steps of pattern formation in the nematode Caenorhabditis elegans. These maternal genes are discussed as belonging to one of three groups. The par group genes establish and maintain polarity in the one-cell zygote in response to sperm entry, defining an anterior/posterior body axis at least in part through interactions with the cyto-skeleton mediated by cortically localized proteins. Blastomere identity group genes act down-stream of the par group to specify the identities of individual embryonic cells, or blastomeres, using both cell autonomous and non-cell autonomous mechanisms. Requirements for the blastomere identity genes are consistent with previous studies suggesting that early asymmetric cleavages in the C. elegans embryo generate six "founder" cells that account for much of the C. elegans body plan. Intermediate group genes, most recently identified, may link the establishment of polarity in the zygote by par group genes to the localization of blastomere identity group gene functions. This review summarizes the known requirements for the members of each group, although it seems clear that additional regulatory genes controlling pattern formation in the early embryo have yet to be identified. An emerging challenge is to link the function of the genes in these three groups into interacting pathways that can account for the specification of the six founder cell identities in the early embryo, five of which produce somatic cell types and one of which produces the germline.

Defective dorsal closure and loss of epidermal decapentaplegic expression in Drosophila fos mutants.

Drosophila kayak mutant embryos exhibit defects in dorsal closure, a morphogenetic cell sheet movement during embryogenesis. Here we show that kayak encodes D-Fos, the Drosophila homologue of the mammalian proto-oncogene product, c-Fos. D-Fos is shown to act in a similar manner to Drosophila Jun: in the cells of the leading edge it is required for the expression of the TGFbeta-like Decapentaplegic (Dpp) protein, which is believed to control the cell shape changes that take place during dorsal closure. Defects observed in mutant embryos, and adults with reduced Fos expression, are reminiscent of phenotypes caused by 'loss of function' mutations in the Drosophila JNKK homologue, hemipterous. These results indicate that D-Fos is required downstream of the Drosophila JNK signal transduction pathway, consistent with a role in heterodimerization with D-Jun, to activate downstream targets such as dpp.

Drosophila Ecdysone Receptor Mutations Reveal Functional Differences among Receptor Isoforms

The steroid hormone ecdysone directs Drosophila metamorphosis via three heterodimeric receptors that differ according to which of three ecdysone receptor isoforms encoded by the EcR gene (EcR-A, EcR-B1, or EcR-B2) is activated by the orphan nuclear receptor USP. We have identified and molecularly mapped two classes of EcR mutations: those specific to EcR-B1 that uncouple metamorphosis, and embryonic-lethal mutations that map to common sequences encoding the DNA- and ligand-binding domains. In the larval salivary gland, loss of EcR-B1 results in loss of activation of ecdysone-induced genes. Comparable transgenic expression of EcR-B1, EcR-B2, and EcR-A in these mutant glands results, respectively, in full, partial, and no repair of that loss.

A limited number of Caenorhabditis elegans genes are readily mutable to dominant, temperature-sensitive maternal-effect embryonic lethality.

Dominant gain-of-function mutations can give unique insights into the study of gene function. In addition, gain-of-function mutations, unlike loss-of-function alleles, are not biased against the identification of genetically redundant loci. To identify novel genetic functions active during Caenorhabditis elegans embryogenesis, we have collected a set of dominant temperature-sensitive maternal-effect embryonic lethal mutations. In a previous screen, we isolated eight such mutations, distributed among six genes. In the present study, we describe eight new dominant mutations that identify only three additional genes, yielding a total of 16 dominant mutations found in nine genes. Therefore, it appears that a limited number of C. elegans genes mutate to this phenotype at appreciable frequencies. Five of the genes that we identified by dominant mutations have loss-of-function alleles. Two of these genes may lack loss-of-function phenotypes, indicating that they are nonessential and so may represent redundant loci. Loss-of-function mutations of three other genes are associated with recessive lethality, indicating nonredundancy.

Mutations resulting in transient and localized degeneration in the developing zebrafish brain

In a large-scale mutagenesis screen in the zebrafish, Danio rerio, we have identified a heterogeneous group of 30 recessive, embryonic lethal mutations characterized by degeneration in the developing central nervous system that is either transient or initially localized to one area of the brain. Transient degeneration is defined as abnormal cell death occurring during a restricted period of development. Following degeneration, the affected structures do not appear to regenerate. In each case degeneration is identified after somitogenesis is complete and is not associated with visually identified patterning defects. These 30 mutations, forming 21 complementation groups, have been classified into four phenotypic groups: group 1, transient degeneration (13 mutations); group 2, spreading degeneration, early onset, in which degeneration is initially confined to the optic tectum but subsequently spreads to other areas of the central nervous system (7 mutations); group 3, late-onset degeneration, initially identified after 4 days (6 mutations); and group 4, degeneration with abnormal pigmentation (4 mutations). Although apoptotic cells are seen in the retina and tectum of all mutants, the distribution, temporal progression, and severity of degeneration vary between mutations. Several mutations also show pleiotropic effects, with degeneration involving extraneural structures including the pharyngeal arches and pectoral fins. We discuss some of the pathways important for cell survival in the nervous system and suggest that these mutations will provide entry points for identifying genes that affect the survival of restricted neural populations.

A Novel KH-Domain Protein Mediates Cell Adhesion Processes inDrosophila

Adhesion of cells to one another and to extracellular matrices has major roles in morphogenetic processes during development. One important family of cell adhesion receptors are the integrins, which inDrosophilahave crucial functions in at least two adhesion-mediated developmental events: embryonic muscle attachment and adhesion of the wing epithelia. We have cloned and characterized a gene (struthio) that is expressed in embryonic mesodermal and muscle cells, including cardioblasts, and epidermal muscle attachment sites in a pattern that is reminiscent of the expression pattern of the PS integrins. Maternal and zygotic transcripts are produced by this gene and encode similar proteins with two alternative carboxy tails. Both proteins contain identical KH domains, a protein sequence motif that is found in numerous proteins that interact with RNA. Thestruthioprotein is highly homologous in a region including the KH domain to the mousequakingandC. elegans gld-1proteins, two developmentally important genes. Somatic homozygous clones of an embryonic lethal mutation in this gene (stru1A122) cause wing blisters and flight impairment, phenotypes which are associated with PS integrin subunit mutations. Thus, thestruthiogene encodes a putative RNA-binding protein that appears to regulate some aspects ofDrosophilaintegrin functioning.

Distinct requirements for somatic and germline expression of a generally expressed Caernorhabditis elegans gene.

In screening for embryonic-lethal mutations in Caenorhabditis elegans, we defined an essential gene (let-858) that encodes a nuclear protein rich in acidic and basic residues. We have named this product nucampholin. Closely homologous sequences in yeast, plants, and mammals demonstrate strong evolutionary conservation in eukaryotes. Nucampholin resides in all nuclei of C. elegans and is essential in early development and in differentiating tissue. Antisense-mediated depletion of LET-858 activity in early embryos causes a lethal phenotype similar to characterized treatments blocking embryonic gene expression. Using transgene-rescue, we demonstrated the additional requirement for let-858 in the larval germline. The broad requirements allowed investigation of soma-germline differences in gene expression. When introduced into standard transgene arrays, let-858 (like many other C. elegans genes) functions well in soma but poorly in germline. We observed incremental silencing of simple let-858 arrays in the first few generations following transformation and hypothesized that silencing might reflect recognition of arrays as repetitive or heterochromatin-like. To give the transgene a more physiological context, we included an excess of random genomic fragments with the injected DNA. The resulting transgenes show robust expression in both germline and soma. Our results suggest the possibility of concerted mechanisms for silencing unwanted germiline expression of repetitive sequences.

Mutations in the Drosophila neuroglian cell adhesion molecule affect motor neuron pathfinding and peripheral nervous system patterning

We have identified and characterized three embryonic lethal mutations that alter or abolish expression of Drosophila Neuroglian and have used these mutations to analyze Neuroglian function during development. Neuroglian is a member of the immunoglobulin superfamily. It is expressed by a variety of cell types during embryonic development, including expression on motoneurons and the muscle cells that they innervate. Examination of the nervous systems of neuroglian mutant embryos reveals that motoneurons have altered pathfinding trajectories. Additionally, the sensory cell bodies of the peripheral nervous system display altered morphology and patterning. Using a temperature-sensitive mutation, the phenocritical period for Neuroglian function was determined to occur during late embryogenesis, an interval which coincides with the period during which neuromuscular connections and the peripheral nervous system pattern are established. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 325–340, 1997

Mutations in theDrosophila neuroglian cell adhesion molecule affect motor neuron pathfinding and peripheral nervous system patterning

We have identified and characterized three embryonic lethal mutations that alter or abolish expression of Drosophila Neuroglian and have used these mutations to analyze Neuroglian function during development. Neuroglian is a member of the immunoglobulin superfamily. It is expressed by a variety of cell types during embryonic development, including expression on motoneurons and the muscle cells that they innervate. Examination of the nervous systems of neuroglian mutant embryos reveals that motoneurons have altered pathfinding trajectories. Additionally, the sensory cell bodies of the peripheral nervous system display altered morphology and patterning. Using a temperature-sensitive mutation, the phenocritical period for Neuroglian function was determined to occur during late embryogenesis, an interval which coincides with the period during which neuromuscular connections and the peripheral nervous system pattern are established.

Mapping of a major genetic modifier of embryonic lethality in TGF beta 1 knockout mice.

The transforming growth factor beta 1 (TGF beta 1) signalling pathway is important in embryogenesis and has been implicated in hereditary haemorrhagic telangiectasia (HHT), atherosclerosis, tumorigenesis and immunomodulation. Therefore, identification of factors which modulate TGF beta 1 bioactivity in vivo is important. On a mixed genetic background, approximately 50% Tgfb1-/- conceptuses die midgestation from defective yolk sac vasculogenesis. The other half are developmentally normal but die three weeks postpartum. Intriguingly, the vascular defects of Tgfb1-/- mice share histological similarities to lesions seen in HHT patients. It has been suggested that dichotomy in Tgfb1-/- lethal phenotypes is due to maternal TGF beta 1 rescue of some, but not all, Tgfb1-/- embryos12. Here we show that the Tgfb1-/- phenotype depends on the genetic background of the conceptus. In NIH/Ola, C57BL/6J/Ola and F1 conceptuses, Tgfb1-/- lethality can be categorized into three developmental classes. A major codominant modifier gene of embryo lethality was mapped to proximal mouse chromosome 5, using a genome scan for non-mendelian distribution of alleles in Tgfb1-/- neonatal animals which survive prenatal lethality. This gene accounts for around three quarters of the genetic effect between mouse strains and can, in part, explain the distribution of the three lethal phenotypes. This approach, using neonatal DNA samples, is generally applicable to identification of loci that influence the effect of early embryonic lethal mutations, thus furthering knowledge of genetic interactions that occur during early mammalian development in vivo.

Mutations affecting cell fates and cellular rearrangements during gastrulation in zebrafish.

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.

Mutations affecting neural survival in the zebrafish Danio rerio

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.

Isolation of T-DNA flanking plant DNA from T-DNA insertional embryo-lethal mutants of Arabidopsis thaliana by plasmid rescue technique

Three T-DNA insertional embryonic lethal mutants from NASC (The Nottingham Arabidopsis Stock Center) were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion. The N4081 mutant has a segregation ratio of 1:3.04 in average and one T-DNA insertion site according to our assay. It was therefore chosen for further analysis. To isolate the joint fragment of T-DNA and plant DNA, the plasmid rescue technique was used. pEL-7, one of plasmids from left border of T-DNA, which contained pBR322 was selected from ampicillin plate. The T-DNA fragment of pEL-7 was checked by restriction enzyme analysis and Southern Blot. Restriction analysis confirmed the presence of known sites of EcoRI, PstI and PvuII on it. For confirming the presence of flanking plant DNA in this plasmid, pEL-7 DNA was labeled and hybridized with wild type and mutant plant DNA. The Southern Blot indicated the hybridization band in both of them. Furthermore, the junction of T-DNA/plant DNA was subcloned into bluescript SK+ and sequenced by Applied Biosystem 373A Sequencer. The results showed the 822 bp fragment contained a 274 bp sequence, which is 99.6% homolog (273bp/274 bp) to Ti plasmid pTi 15955 DNA. Ten bp of left 25 bp border repeat were also found in the juction of T-DNA and Plant DNA. Taken together, pEL-7 should contain a joint fragment of T-DNA and flanking plant DNA. This plasmid DNA could be used for the isolation of plant gene, which will be helpful to elucidate the relationship between gene function and plant embryo development.

Altered development of spinal cord in the mouse mutant ( Patch) lacking the PDGF receptor α-subunit gene

The platelet-derived growth factor receptor α subunit (PDGFRα) is expressed by glial precursors, glial cells, and some peripheral neurons during normal rodent development. Its ligands are expressed ubiquitously in neurons, including sensory and motor neurons. Thus, neuronally secreted PDGF-A may play a paracrine role in the development of both glial cells and peripheral neurons. The Patch ( Ph) mutation, which is a deletion of the PDGFRα, is a homozygous embryonic lethal mutation in the mouse. Previously, several developmental abnormalities, including deficiencies in connective tissues in many organs, aberrant neural crest cell migration, and defects in non-neuronal derivatives of crest cells, have been shown to be associated with the Patch mutation. However, whether and the extent to which motor and sensory neurons are affected by the mutation are not known. Here, we have examined the survival and/or morphological differentiation of spinal motor and sensory (dorsal root ganglion) neurons during the period of naturally occurring cell death, i.e., between E14 and E18, in control and Ph/Ph mice. The results show a 65–70% decrease in motor and sensory neuron numbers in Ph/Ph mice, compared to controls, at all stages examined. Furthermore, motoneurons in Ph/Ph mice were significantly smaller than those in controls. Because of the bidirectional nature of neuron-glial cell interactions, these results suggest that PDGFR α plays an important role in glial cell development and, thus, indirectly in neuronal cell development or, alternatively, that PDGF and the PDGFR α are directly involved in peripheral neuron survival and development by an autocrine/paracrine mechanism.

Synthesis of a New Substrate for Detection of lacZ Gene Expression in Live Drosophila Embryos

Escherichia coli lacZ, which encodes beta-galactosidase, has become a widely used reporter gene to study the developmental regulation of gene expression in a variety of organisms. To detect the presence of the beta-galactosidase, the sample must be fixed and appropriately stained. This sort of analysis yields rather crude estimates of the spatial-temporal changes in gene expression patterns. In addition, one cannot recover interesting specimens for propagation. A novel fluorogenic beta-galactosidase substrate for use in live Drosophila melanogaster embryos has been designed and synthesized. This compound provides a means to determine gene dosage in live embryos so that one can unambiguously determine the genotype of a living embryo. This will be useful for detailed analysis of cellular and morphogenetic behavior changes in live embryos that are homozygous for embryonic lethal mutations. In the course of testing this compound, a new beta-galactoside hydrolytic activity, different from the previously identified beta-galactosidase, has been discovered to reside in macrophages and the intervitelline space.

Morphology of embryos and constitution of yolk proteins in an embryonic lethal mutant, Df(14)Nl, of Bombyx mori

Morphology of embryos and constitution of yolk proteins in an embryonic lethal mutant, Df(14)Nl, of Bombyx mori