An expanded role for the TWN1 gene in embryogenesis: defects in cotyledon pattern and morphology in the twn1 mutant of Arabidopsis (Brassicaceae)

The suspensor is a specialized basal structure that differentiates early in plant embryogenesis to support development of the embryo proper. Suspensor differentiation in Arabidopsis is maintained in part by the TWIN1 (TWN1) gene, which suppresses embryogenic development in suspensor cells: twn1 mutants produce supernumerary embryos via suspensor transformation. To better understand mechanisms of suspensor development and further investigate the function of TWN1, we have characterized late-embryo and post-embryonic development in the twn1 mutant, using seedling culture, microscopy, and genetics. We report here that the twn1 mutation disrupts cotyledon number, arrangement, and morphology and occasionally causes partial conversion of cotyledons into leaves. These defects are not a consequence of suspensor transformation. Thus, in addition to its basal role in suspensor differentiation, TWN1 influences apical pattern and morphology in the embryo proper. To determine whether other genes can similarly affect both suspensor and cotyledon development, we looked for twinning in Arabidopsis mutants previously identified by their abnormal cotyledon phenotypes. One such mutant, amp1, produced a low frequency of twin embryos by suspensor transformation. Our results suggest that mechanisms that maintain suspensor identity also function later in development to influence organ formation at the embryonic shoot apex. We propose that TWN1 functions in cell communication pathways that convey local positional information in both the apical and basal regions of the Arabidopsis embryo.     

Three-dimensional analysis of vascular development in the mouse embryo

Key vasculogenic (de-novo vessel forming) and angiogenic (vessel remodelling) events occur in the mouse embryo between embryonic days (E) 8.0 and 10.0 of gestation, during which time the vasculature develops from a simple circulatory loop into a complex, fine structured, three-dimensional organ. Interpretation of vascular phenotypes exhibited by signalling pathway mutants has historically been hindered by an inability to comprehensively image the normal sequence of events that shape the basic architecture of the early mouse vascular system. We have employed Optical Projection Tomography (OPT) using frequency distance relationship (FDR)-based deconvolution to image embryos immunostained with the endothelial specific marker PECAM-1 to create a high resolution, three-dimensional atlas of mouse vascular development between E8.0 and E10.0 (5 to 30 somites). Analysis of the atlas has provided significant new information regarding normal development of intersomitic vessels, the perineural vascular plexus, the cephalic plexus and vessels connecting the embryonic and extraembryonic circulation. We describe examples of vascular remodelling that provide new insight into the mechanisms of sprouting angiogenesis, vascular guidance cues and artery/vein identity that directly relate to phenotypes observed in mouse mutants affecting vascular development between E8.0 and E10.0. This atlas is freely available at http://www.mouseimaging.ca/research/mouse_atlas.html and will serve as a platform to provide insight into normal and abnormal vascular development.     

Isolation and Characterization of 51 embryo-specific Mutations of Maize

A plant embryo consists of an embryonic axis, which eventually grows into the adult body, and one or two nutritive structures, the cotyledons. In the grasses embryo morphogenesis can be divided into three periods: during the first the embryo is regionalized into an embryo proper and suspensor, during the second the embryonic axis is established, and during the third vegetative structures are elaborated. Maize, with its well-characterized embryo-genesis, powerful genetics, and transposon tagging stocks, offers an attractive system for mutational analysis of these events. We have isolated 51 embryo-specific (emb) mutations from active Robertson's Mutator maize stocks. These are single-gene recessive lethals that represent at least 45 independent mutation events. Each of the 25 mutations was located to a chromosome arm using a B-A translocation set that uncovers approximately 40% of the genome; the same test failed to locate 20 others. The embryo phenotype of 27 mutations was characterized by examining mature mutant embryos in fresh dissection: the various emb mutations differ in phenotype and each is consistent in its expression. All 27 mutations result in retarded embryos that are morphologically abnormal. Nine mutants are blocked during the first period; 10 mutants are blocked during the second period; and eight mutants are blocked during the third period. Based on both the genetic and developmental data, it is likely that there are many loci that can mutate to give the emb phenotype and that these genes are crucial to the morphogenesis of the embryo.     

Developmental mutants showing abnormal organ differentiation in rice embryos

Summary Zygotes of rice (Oryza sativa L. cv Taichung 65) were treated with 1.0 mM solution of the chemical mutagen N-methyl-N-nitrosourea. Out of 1420 M₂ lines, 28 single-locus recessive mutants on embryogenesis were identified. Among them, we analyzed 11 mutants in the present study, which differentiated the shoot (plumule) and/or root (radicle) with abnormality. Of the 11 mutants, two showed no shoot differentiation with normal root. On the other hand, we could not detect any mutant which exhibited a normal shoot without a root. This suggests that shoot and root are genetically controlled by different loci and that the alleles associated with shoot formation mutate more frequently than do those of the root. Five mutants showed aberrant morphology of shoot when both the shoot and root developed. One of them, odm 5 (organ differententiation mutant 5) was germinable, but produced many fine and twisted leaves. This mutant was, however, lethal at the early post-germination stage under the usual cultural conditions. In another mutant (odm 4), shoot differentiation seemed to be initiated at an arbitrary position, resulting in a very abnormal morphology of the shoot when the position fronted the endosperm. The other two mutants showed abnormal morphology of both the shoot and root. One (odm 11) of the remaining two mutants showed a wide variation of abnormalities including no organ differentiation, either shoot or root differentiation and the development of both shoot and root with abnormalities. The last one (odm 16) was unique. It had an embryo with normal shoot and root but the embryo size was only one-third to one-half of normal embryos in length. Of course, the shoot and root are also small but viable. Therefore, odm 16 is considered to be a mutant in the size regulation of the embryo. Although an allelism test has not yet been done, most of these mutants are probably non-allelic, as the phenotypic abnormality differs largely with each one. In rice, the shoot and root highly differentiate in contrast to dicotyledonous embryo. Accordingly, these developmental mutants are very useful materials for investigating the regulatory mechanism of gene expression in organ differentiation.     

An F1 genetic screen for maternal-effect mutations affecting embryonic pattern formation in Drosophila melanogaster

Large-scale screens for female-sterile mutations have revealed genes required maternally for establishment of the body axes in the Drosophila embryo. Although it is likely that the majority of components involved in axis formation have been identified by this approach, certain genes have escaped detection. This may be due to (1) incomplete saturation of the screens for female-sterile mutations and (2) genes with essential functions in zygotic development that mutate to lethality, precluding their identification as female-sterile mutations. To overcome these limitations, we performed a genetic mosaic screen aimed at identifying new maternal genes required for early embryonic patterning, including zygotically required ones. Using the Flp-FRT technique and a visible germline clone marker, we developed a system that allows efficient screening for maternal-effect phenotypes after only one generation of breeding, rather than after the three generations required for classic female-sterile screens. We identified 232 mutants showing various defects in embryonic pattern or morphogenesis. The mutants were ordered into 10 different phenotypic classes. A total of 174 mutants were assigned to 86 complementation groups with two alleles on average. Mutations in 45 complementation groups represent most previously known maternal genes, while 41 complementation groups represent new loci, including several involved in dorsoventral, anterior-posterior, and terminal patterning.     

Hierarchical regulation of organ differentiation during embryogenesis in rice

In plants, genetic mechanisms leading to shoot and root formation are almost unknown. Because basic body organization of such organisms is established during embryogenesis, induction and isolation of embryonic mutants is a promising approach to the study of plant development. The study of available embryonic mutants of rice indicates the existence of three major developmental processes taking place during embryogenesis before morphogenetic events start: determination of organ differentiation, positional regulation of organs and size regulation of the embryo. The consideration of specific rice mutants supports the existence of two types of mutations in each regulatory process, one affecting the embryo as a whole and the second concerning more restricted embryonal regions. A hierarchical type of control of rice embryogenesis is suggested.     

Zebrafish endzone regulates neural crest-derived chromatophore differentiation and morphology

The development of neural crest-derived pigment cells has been studied extensively as a model for cellular differentiation, disease and environmental adaptation. Neural crest-derived chromatophores in the zebrafish (Danio rerio) consist of three types: melanophores, xanthophores and iridiphores. We have identified the zebrafish mutant endzone (enz), that was isolated in a screen for mutants with neural crest development phenotypes, based on an abnormal melanophore pattern. We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced. Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size. We demonstrate that enz function is required cell autonomously by melanophores and that the enz locus is located on chromosome 7. In addition, zebrafish enz appears to selectively regulate chromatophore development within the neural crest lineage since all other major derivatives develop normally. Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology. Thus, although developmental regulation of different chromatophore sublineages in zebrafish is in part genetically distinct, enz provides an example of a common regulator of neural crest-derived chromatophore differentiation and morphology.     

Multiple, distinct isoforms of sucrose synthase in pea

Genes encoding three isoforms of sucrose synthase (Sus1, Sus2, and Sus3) have been cloned from pea (Pisum sativum). The genes have distinct patterns of expression in different organs of the plant, and during organ development. Studies of the isoforms expressed as recombinant proteins in Escherichia coli show that they differ in kinetic properties. Although not of great magnitude, the differences in properties are consistent with some differentiation of physiological function between the isoforms. Evidence for differentiation of function in vivo comes from the phenotypes of rug4 mutants of pea, which carry mutations in the gene encoding Sus1. One mutant line (rug4-c) lacks detectable Sus1 protein in both the soluble and membrane-associated fractions of the embryo, and Sus activity in the embryo is reduced by 95%. The starch content of the embryo is reduced by 30%, but the cellulose content is unaffected. The results imply that different isoforms of Sus may channel carbon from sucrose towards different metabolic fates within the cell.     

Mutational analysis of morphogenesis of the maize embryo

Morphogenesis of the maize embryo is controlled by many genes. A group of 51 embryo-specific (emb) mutations representing at least 45 independent mutation events and many different gene loci have been isolated from active Robertson's Mutator stocks. The authors have reported previously that the embryo phenotype of 27 of these mutations, characterized by examining mature embryos in fresh dissection. The maximal development capacity of the 24 emb mutations are reported here which have not been reported previously. All result in retarded embryos that are morphologically abnormal. Three of the mutants are blocked during the first phase of morphogenesis, the period in which the basal-apical asymmetry is established and the embryo is regionalized into suspensor and embryo proper. Nineteen mutants are blocked during the second phase, the period in which radial asymmetry appears, the embryonic axis is established at a different angle than the original basal-apical axis of the zygote and the vegetative organ primordia of the adult plant make their first appearance. Two mutants are blocked or altered during the third phase, the period in which vegetative structures are elaborated. Some of the mutants affected in the first phase of morphogenesis may have defective mechanisms for establishing basal-apical asymmetry, including possibly the asymmetric distribution of morphogenic determinants. Similarly, some of the mutants affected in the second phase may be altered in the mechanisms establishing radial asymmetry and the origin of the meristems. Mutations of the first type may act as early as the first cell division when the zygote undergoes a transverse division, while mutations of the second type are likely to act during the proembryo and transition stages. Both types include mutations affecting embryo pattern formation. Mutations affecting the third phase of morphogenesis may identify genes regulating reiterative morphogenic processes of vegetative plant development and events of embryo maturation. This group of 24 mutations is like that reported previously in representing genes that are crucial to embryo morphogenesis.     

Lrp5 and Lrp6 redundantly control skeletal development in the mouse embryo

The role of Wnt signaling in osteoblastogenesis in the embryo remains to be fully established. Although β-catenin, a multifunctional protein also mediating canonical Wnt signaling, is indispensable for embryonic osteoblast differentiation, the roles of the key Wnt co-receptors Lrp5 and Lrp6 are unclear. Indeed, global deletion of either Lrp5 or Lrp6 did not overtly affect osteoblast differentiation in the mouse embryo. Here, we generated mice lacking both receptors specifically in the embryonic mesenchyme and observed an absence of osteoblasts in the embryo. In addition, the double-deficient embryos developed supernumerary cartilage elements in the zeugopod, revealing an important role for mesenchymal Lrp5/6 signaling in limb patterning. Importantly, the phenotypes of the Lrp5/6 mutant closely resembled those of the β-catenin-deficient embryos. These phenotypes are likely independent of any effect on the adherens junction, as deletion of α-catenin, another component of the complex, did not cause similar defects. Thus, Lrp5 and 6 redundantly control embryonic skeletal development, likely through β-catenin signaling.     

A cytokine secreted from the suboesophageal body is essential for morphogenesis of the insect head

The suboesophageal body of insects was identified over a century ago in the silkworm embryo, but its biological function is still unknown. We discovered that this tissue is differentiated in the earliest embryonic stages of the cabbage armyworm and secretes the insect cytokine, growth-blocking peptide (GBP), transiently from 24 to 60 h after oviposition when gastrulation is in progress. Over-expression of GBP, achieved by microinjection of the GBP gene driven by a cytomegalovirus (CMV) constitutive promoter, resulted in complex deformities of the procephalon (embryonic head). Severe abnormal phenotypes of the head structure were produced by silencing the GBP expression in the embryo by treating with GBP double-stranded RNA: the procephalon-containing optic lobes diminished and completely separated into bilateral halves. This indicates that GBP secreted from the suboesophageal body plays an essential role in the formation of the procephalic domain during early embryogenesis. The cytokine-induced fusion of bilateral procephalic lobes is thought to be evolutionarily conserved at least in insects, because of the widespread occurrence of the suboesophageal body in insect embryos.     

Genetic analysis of functional domains within the Drosophila LARK RNA-binding protein

LARK is an essential Drosophila RNA-binding protein of the RNA recognition motif (RRM) class that functions during embryonic development and for the circadian regulation of adult eclosion. LARK protein contains three consensus RNA-binding domains: two RRM domains and a retroviral-type zinc finger (RTZF). To show that these three structural domains are required for function, we performed a site-directed mutagenesis of the protein. The analysis of various mutations, in vivo, indicates that the RRM domains and the RTZF are required for wild-type LARK functions. RRM1 and RRM2 are essential for viability, although interestingly either domain can suffice for this function. Remarkably, mutation of either RRM2 or the RTZF results in the same spectrum of phenotypes: mutants exhibit reduced viability, abnormal wing and mechanosensory bristle morphology, female sterility, and flightlessness. The severity of these phenotypes is similar in single mutants and double RRM2; RTZF mutants, indicating a lack of additivity for the mutations and suggesting that RRM2 and the RTZF act together, in vivo, to determine LARK function. Finally, we show that mutations in RRM1, RRM2, or the RTZF do not affect the circadian regulation of eclosion, and we discuss possible interpretations of these results. This genetic analysis demonstrates that each of the LARK structural domains functions in vivo and indicates a pleiotropic requirement for both the LARK RRM2 and RTZF domains.     

Conserved Functions of the MATE Transporter BIG EMBRYO1 in Regulation of Lateral Organ Size and Initiation Rate

Genetic networks that determine rates of organ initiation and organ size are key regulators of plant architecture. Whereas several genes that influence the timing of lateral organ initiation have been identified, the regulatory pathways in which these genes operate are poorly understood. Here, we identify a class of genes implicated in regulation of the lateral organ initiation rate. Loss-of-function mutations in the MATE transporter encoded by maize (Zea mays) Big embryo 1 (Bige1) cause accelerated leaf and root initiation as well as enlargement of the embryo scutellum. BIGE1 is localized to trans-Golgi, indicating a possible role in secretion of a signaling molecule. Interestingly, phenotypes of bige1 bear striking similarity to cyp78a mutants identified in diverse plant species. We show that a CYP78A gene is upregulated in bige1 mutant embryos, suggesting a role for BIGE1 in feedback regulation of a CYP78A pathway. We demonstrate that accelerated leaf formation and early flowering phenotypes conditioned by mutants of Arabidopsis thaliana BIGE1 orthologs are complemented by maize Bige1, showing that the BIGE1 transporter has a conserved function in regulation of lateral organ initiation in plants. We propose that BIGE1 is required for transport of an intermediate or product associated with the CYP78A pathway.     

Isolation and complementation of nitrogen fixation mutants of the cyanobacterium Anabaena sp. strain PCC 7120.

Approximately 140 mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on media lacking fixed nitrogen (Fix-) were isolated after mutagenesis with diethyl sulfate and penicillin enrichment. A large cosmid library of wild-type Anabaena sp. strain PCC 7120 DNA was constructed in a mini-RK-2 shuttle vector, and seven mutants representing several morphologically abnormal heterocyst phenotypes were complemented. One of these mutants, 216, failed to differentiate heterocysts. All of these mutants except 216 reduced acetylene under anaerobic conditions, indicating that they are not defective in nitrogen fixation per se. Several cosmids were isolated from each complemented mutant and in most cases showed similar restriction patterns. Comparisons of the complementing cosmids from mutant 216 and two other phenotypically distinct mutants by restriction enzyme analysis identified a common region. This region, when present in either a cosmid or a 9.5-kb NheI subclone, is capable of efficiently complementing all three mutants. A 2.4-kb subclone of this region complements mutant 216 only.     

Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells.

A series of adenovirus type 5 (Ad5) deletion, insertion and substitution mutants, some of which are defective for transformation of rat cells, have been isolated. The mutants were selected as variants which lack the Xba I endonuclease cleavage site at 4 map units on the viral chromosome. The deletions range in size from 150-2300 bp and are located between 1.5 and 10.5 map units. The mutants can be propagated in 293 cells (Ad5-transformed human embryonic kidney cells), but are defective for growth in HeLa or human embryonic kidney cells. No viral DNA synthesis was observed in mutant virus-infected HeLa cells. All but one of the deletion mutants tested were defective for transformation of rat embryo and rat embryo brain cells.     

Morphology and ultrastructure of 11 barley shrunken endosperm mutants

Summary Eleven Na-azide induced barley shrunken endosperm mutants expressing xenia (sex) were characterized genetically and histologically. All mutants have reduced kernel size with kernel weights ranging from 11 to 57% of the wild type. With one exception, the mutant phenotypes are ascribable to single recessive mutant alleles, giving rise to a ratio of 31 of normal and shrunken kernels on heterozygous plants. One mutant (B10), also monofactorially inherited, shows a gene dosage dependent pattern of expression in the endosperm. Among the 8 mutants tested for allelism, no allelic mutant genes were discovered. By means of translocation mapping, the mutant gene of B10 was localized to the short arm of chromosome 7, and that of B9 to the short arm of chromosome 1. Based on microscopy studies, the mutant kernel phenotypes fall into three classes, viz. mutants with both endosperm and embryo affected and with a non-viable embryo, mutants with both endosperm and embryo affected and with a viable embryo giving rise to plants with a clearly mutant phenotype, and finally mutants with only the endosperm affected and with a normal embryo giving rise to plants with normal phenotype. The mutant collection covers mutations in genes participating in all of the developmental phases of the endosperm, i.e. the passage from syncytial to the cellular endosperm, total lack of aleurone cell formation and disturbance in the pattern of aleurone cell formation. In the starchy endosperm, varying degrees of cell differentiation occur, ranging from slight deviations from wild type to complete loss of starchy endosperm traits. In the embryo, blocks in the major developmental phases are represented in the mutant collection, including arrest at the proembryo stage, continued cell divisions but no differentiation, and embryos deviating only slightly from the wild type.