Effects of mechanical stretching of embryonic tissues on axial structure formation in <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Effect of mechanical stretch on the differentiation of axial anlages and Chordin gene expression was studied in sandwich explants prepared from embryonic tissues of Xenopus laevis at the early gastrula stage in two variants: with dissected or intact dorso-medial region. In the first case, convergent cell movements were suppressed and properly organized axial organs (notochord and somites) were almost completely absent. However, they developed if the explants of such type were artificially stretched in the ventro-dorsal direction. In this case, axial organs elongated in the line of stretching, that is in the direction vertical to their normal orientation. Segmented mesoderm was always in contact with the chord anlage. In situ hybridization revealed that the area of Chordin gene expression was also extended in the direction of stretching. PCR showed that Chordin gene expression in stretched explants with disrupted dorso-medial region was statistically at the same level as in the explants with intact dorso-medial region. At the same time, the corresponding gene expression in unstretched explants with disrupted dorso-medial region was statistically higher. The obtained data indicate that mechanical stretch and associated cell movements are a necessary and sufficient condition for the formation of proper histological structure of axial organs and regulation of Chordin gene expression.     

The E3 ubiquitin ligase Hace1 is required for early embryonic development in <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Background

HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1) regulates a wide variety of cellular processes. It has been shown that one of the targets of HACE1 is the GTP-bound form of the small GTPase Rac1. However, the role of HACE1 in early development remains unknown.

Results

In situ hybridization revealed that Xenopus laevis hace1 is specifically expressed in the ectoderm at the blastula and gastrula stages and in the epidermis, branchial arch, kidney, and central nervous system at the tailbud stage. Knockdown of hace1 in Xenopus laevis embryos via antisense morpholino oligonucleotides led to defects in body axis elongation, pigment formation, and eye formation at the tadpole stage. Experiments with Keller sandwich explants showed that hace1 knockdown inhibited convergent extension, a morphogenetic movement known to be crucial for body axis elongation. In addition, time lapse imaging of whole embryos during the neurula stage indicated that hace1 knockdown also delayed neural tube closure. The defects caused by hace1 knockdown were partly rescued by knockdown of rac1. Moreover, embryos expressing a constitutively active form of Rac1 displayed phenotypes similar to those of embryos with hace1 knocked down.

Conclusions

Our results suggest that Xenopus laevis hace1 plays an important role in early embryonic development, possibly via regulation of Rac1 activity.

     

Role of cooperative cell movements and mechano-geometric constrains in patterning of axial rudiments in <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryos

The role of cooperative cell movements has been explored in establishment of regular segregation of the marginal zone of Xenopus laevis embryos into the main axial rudiments: notochord, somites and neural tissue. For this purpose, the following operations were performed at the late blastula-early gastrula stages: (1) isolation of marginal zones, (2) addition of the ventral zone fragments to the marginal zones, (3) dissection of isolated marginal zones along either ventral (a) or dorsal (b) midlines, (4) immediate retransplantation of excised fragments of the suprablastoporal area to the same places without rotation or after 90° rotation, (5) Π-shaped separation of the suprablastoporal area either anteriorly or posteriorly. In experiments 1, 4, and 5, lateromedial convergent cell movements and differentiation of the axial rudiments were suppressed. In experiments 4 and 5, cell movements were reoriented ventrally, the entire embryo architecture was extensively reconstructed, and the axial rudiments were relocated to the blastopore lateral lips. In experiment 3, convergent cell movements were restored and oriented either towards the presumptive embryo midline (a), or in the perpendicular direction (b). In both cases, well developed axial rudiments elongated perpendicularly to cell convergences were formed. If the areas of axial rudiment formation were curved, mesodermal somites and neural tissue were always located on the convex (stretched) and concave (compressed) sides, respectively. We conclude that no stable prepatterning of the marginal zone takes place until at least the midgastrula stage. This prepatterning requires cooperative cell movements and associated mechano-geometric constrains.     

Cilia are required for asymmetric <Emphasis Type="Italic">nodal</Emphasis> induction in the sea urchin embryo

Background

Left-right (LR) organ asymmetries are a common feature of metazoan animals. In many cases, laterality is established by a conserved asymmetric Nodal signaling cascade during embryogenesis. In most vertebrates, asymmetric nodal induction results from a cilia-driven leftward fluid flow at the left-right organizer (LRO), a ciliated epithelium present during gastrula/neurula stages. Conservation of LRO and flow beyond the vertebrates has not been reported yet.

Results

Here we study sea urchin embryos, which use nodal to establish larval LR asymmetry as well. Cilia were found in the archenteron of embryos undergoing gastrulation. Expression of foxj1 and dnah9 suggested that archenteron cilia were motile. Cilia were polarized to the posterior pole of cells, a prerequisite of directed flow. High-speed videography revealed rotating cilia in the archenteron slightly before asymmetric nodal induction. Removal of cilia through brief high salt treatments resulted in aberrant patterns of nodal expression. Our data demonstrate that cilia - like in vertebrates - are required for asymmetric nodal induction in sea urchin embryos.

Conclusions

Based on these results we argue that the anterior archenteron represents a bona fide LRO and propose that cilia-based symmetry breakage is a synapomorphy of the deuterostomes.

     

Topographic changes in nascent and early mesoderm in amphioxus embryos studied by DiI labeling and by in situ hybridization for a Brachyury gene

 In amphioxus embryos, the nascent and early mesoderm (including chorda-mesoderm) was visualized by expression of a Brachyury gene (AmBra-2). A band of mesoderm is first detected encircling the earliest (vegetal plate stage) gastrula sub-equatorially. Soon thereafter, the vegetal plate invaginates, resulting in a cap-shaped gastrula with the mesoderm localized at the blastoporal lip and completely encircling the blastopore. As the gastrula stage progresses, DiI (a vital dye) labeling demonstrates that the entire mesoderm is internalized by a slight involution of the epiblast into the hypoblast all around the perimeter of the blastopore. Subsequently, during the early neurula stage, the internalized mesoderm undergoes anterior extension mid-dorsally (as notochord) and dorsolaterally (in paraxial regions where segments will later form). By the late neurula stage, AmBra-2 is no longer transcribed throughout the mesoderm as a whole; instead, expression is detectable only in the posterior mesoderm and in the notochord, but not in paraxial mesoderm where definitive somites have formed. Received: 28 November 1996 / Accepted: 2 January 1997     

Nodal and BMP expression during the transition to pentamery in the sea urchin <Emphasis Type="Italic">Heliocidaris erythrogramma</Emphasis>: insights into patterning the enigmatic echinoderm body plan

Background

The molecular mechanisms underlying the development of the unusual echinoderm pentameral body plan and their likeness to mechanisms underlying the development of the bilateral plans of other deuterostomes are of interest in tracing body plan evolution. In this first study of the spatial expression of genes associated with Nodal and BMP2/4 signalling during the transition to pentamery in sea urchins, we investigate Heliocidaris erythrogramma, a species that provides access to the developing adult rudiment within days of fertilization.

Results

BMP2/4, and the putative downstream genes, Six1/2, Eya, Tbx2/3 and Msx were expressed in the earliest morphological manifestation of pentamery during development, the five hydrocoele lobes. The formation of the vestibular ectoderm, the specialized region overlying the left coelom that forms adult ectoderm, involved the expression of putative Nodal target genes Chordin, Gsc and BMP2/4 and putative BMP2/4 target genes Dlx, Msx and Tbx. The expression of Nodal, Lefty and Pitx2 in the right ectoderm, and Pitx2 in the right coelom, was as previously observed in other sea urchins.

Conclusion

That genes associated with Nodal and BMP2/4 signalling are expressed in the hydrocoele lobes, indicates that they have a role in the developmental transition to pentamery, contributing to our understanding of how the most unusual body plan in the Bilateria may have evolved. We suggest that the Nodal and BMP2/4 signalling cascades might have been duplicated or split during the evolution to pentamery.

     

Different response of an elite <Emphasis Type="Italic">Bt</Emphasis> restorer line of hybrid rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) in adaptation to nitrogen deficiency

Transgenic Bacillus thuringiensis (Bt) rice have been reported to acquire effective resistance against the target pests; however, the insertion and expression of alien Bt genes may have some unintended effects on the growth characteristics of rice. A screen-house experiment was conducted and repeated twice to investigate the growth characteristics and Bt protein expressions in two Bt rice lines [MH63 (Cry2A*) and MH63 (Cry1Ab/Ac)], which had different Bt protein expression levels in leaves, under zero nitrogen (N0) and recommended nitrogen (NR) fertilizer applications. Compared to the counterpart MH63, MH63 (Cry2A*) under N0 experienced accelerated leaf senescence and a lower internal N use efficiency (IE_N), resulting in a 23.2% decrease in grain yield and a lower accumulated biomass. These variations were revealed to be correlated to the higher ratio of the Bt protein content to the soluble protein content (BTC/SPC) with a maximum value of 4.3‰ in MH63 (Cry2A*) leaves in the late growth stage. Under NR, no differences in growth characteristics between MH63 (Cry2A*) and MH63 were found. The growth characteristics of MH63 (Cry1Ab/Ac), with a lower BTC/SPC in the late growth stage compared to MH63 (Cry2A*), were identical to those of MH63 under the two N applications. Results show that the transgenic Bt rice MH63 (Cry2A*), with a relatively higher Bt protein expression in the late growth stage, had an inferior adaptation to nitrogen deficiency compared to its non-Bt counterpart. And this inferior adaptation was found to be correlated with the higher BTC/SPC in MH63 (Cry2A*) leaves in the late growth stage.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of two <Emphasis Type="Italic">Populus</Emphasis> hybrid clones. The role of pectin domains in cell processes underlying shoot organogenesis induction

An efficient plant regeneration protocol has been established for two commercial Populus hybrid clones, MC (Populus × euramericana) and UNAL (Populus × interamericana). The culture of internode segments on Murashige and Skoog (MS) medium with 0.5 μM α-naphthalene acetic acid (NAA) and 4 μM N⁶-benzyladenine for 7 weeks (2 weeks in absence of activated charcoal and 5 weeks in its presence) resulted in the highest frequency of shoot regeneration (100 % for MC and 82 % for UNAL). All regenerated shoots longer than 2 cm rooted on half-strength MS medium, independent of the addition of 0.1 μM NAA. Nevertheless, shoots developed better-formed roots in NAA-free medium, which had a positive effect on the acclimatization of plants. In order to know the cellular processes underlying in vitro shoot organogenesis, a histological study was made in UNAL internode-explants. Results revealed that in vitro culture caused swelling around the cut-off zones in all explants, but only those undergoing organogenesis formed proliferation centers under subepidermal cells, which led to formation of bud primordia. Moreover, in vivo tissues and explants with different in vitro response showed different immunolabelling patterns when they were treated with fluorescentmonoclonal antibodies directed to several pectin-polysaccharides of the cell wall. Results allow us to assign a predominant role of homogalacturonan with a low degree of methyl-esterification in the initiation of bud primordia, a role of β-1,4-D-galactan side chains of rhamnogalacturonan-I in the cellular differentiation, ra ole of α-1,5-L-arabinan side chains of rhamnogalacturonan-I and of homogalacturonan with a high degree of methyl-esterification in cell division and growth.     

Artificial transfer of a thelytoky-inducing <Emphasis Type="Italic">Wolbachia</Emphasis> endosymbiont between strains of the endoparasitoid wasp <Emphasis Type="Italic">Asobara japonica</Emphasis> (Hymenoptera: Braconidae)

Infection with Wolbachia is known to induce diploidization of haploid eggs and enables the production of females from unfertilized eggs. Although there have been several attempts to achieve the artificial horizontal transfer of thelytoky-inducing Wolbachia in parasitoid wasps, the artificial induction of thelytoky has generally been unsuccessful. In this study, we used two strains of Asobara japonica as study materials—one infected with thelytoky-inducing Wolbachia and the other not. We investigated methods of artificially inducing thelytoky by transferring thelytoky-inducing Wolbachia from wasps of the infected strain (the donor wasps) to wasps that had been cured of Wolbachia and to wasps of the uninfected strain (the recipient wasps). To examine the efficiencies of various methods of transfection, we compared the survival and infection rates of recipient wasps that received microinjections at the pupal and adult stages and in different body parts. We also examined the infection rate of the recipients due to cannibalism of Wolbachia-infected pupae. Among those methods, only microinjection at the adult stage resulted in the successful artificial horizontal transfer of Wolbachia, and some of the Wolbachia-infected wasps showed incomplete thelytoky. A low Wolbachia titer in the artificially infected wasps may explain why the thelytoky was incomplete.     

Characterization of an Alpha Type Carbonic Anhydrase from <Emphasis Type="Italic">Paracentrotus lividus</Emphasis> Sea Urchin Embryos

Carbonic anhydrases (CA) are zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the sea urchin, CA has a role in the formation of the calcitic skeleton during embryo development. Here, we report a newly identified mRNA sequence from embryos of the sea urchin Paracentrotus lividus, referred to as Pl-can. The complete coding sequence was identified with the aid of both EST databases and experimental procedures. Pl-CAN is a 447 aa-long protein, with an estimated molecular mass of 48.5 kDa and an isoelectric point of 6.83. The in silico study of functional domains showed, in addition to the alpha type CA-specific domain, the presence of an unexpected glycine-rich region at the N-terminal of the molecule. This is not found in any other species described so far, but probably it is restricted to the sea urchins. The phylogenetic analysis indicated that Pl-CAN is evolutionarily closer to human among chordates than to other species. The putative role(s) of the identified domains is discussed. The Pl-can temporal and spatial expression profiles, analyzed throughout embryo development by comparative qPCR and whole-mount in situ hybridization (WMISH), showed that Pl-can mRNA is specifically expressed in the primary mesenchyme cells (PMC) of the embryo and levels increase along with the growth of the embryonic skeleton, reaching a peak at the pluteus stage. A recombinant fusion protein was produced in E. coli and used to raise specific antibodies in mice recognized the endogenous Pl-CAN by Western blot in embryo extracts from gastrula and pluteus.     

Genotype independent and efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of the medicinal plant <Emphasis Type="Italic">Withania somnifera</Emphasis> Dunal

Withania somnifera one of the most reputed Indian medicinal plant has been extensively used in traditional and modern medicines as active constituents. A high frequency genotype and chemotype independent Agrobacterium-mediated transformation protocol has been developed for W. somnifera by optimizing several factors which influence T-DNA delivery. Leaf and node explants of Withania chemotype was transformed with A. tumefaciens strain GV3101 harboring pIG121Hm plasmid containing the gusA gene encoding β-glucuronidase (GUS) as a reporter gene and the hptII and the nptII gene as selection markers. Various factors affecting transformation efficiency were optimized; as 2 days preconditioning of explants on MS basal supplemented with TDZ 1 μM, Agrobacterium density at OD₆₀₀ 0.4 with inclusion of 100 μM acetosyringone (As) for 20 min co-inoculation duration with 48 h of co-cultivation period at 22 °C using node explants was found optimal to improved the number of GUS foci per responding explant from 36?±?13.2 to 277.6?±?22.0, as determined by histochemical GUS assay. The PCR and Southern blot results showed the genomic integration of transgene in Withania genome. On average basis 11 T₀ transgenic plants were generated from 100 co-cultivated node explants, representing 10.6 % transformation frequency. Our results demonstrate high frequency, efficient and rapid transformation system for further genetic manipulation in Withania for producing engineered transgenic Withania shoots within very short duration of 3 months.     

Morphological and genetic characterization of a new cytoplasmic male sterility system (<Emphasis Type="Italic">oxa</Emphasis> CMS) in stem mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>)

Key message

oxa CMS is a new cytoplasmic male sterility type in Brassica juncea.

Abstract

oxa CMS is a cytoplasmic male sterility (CMS) line that has been widely used in the production and cultivation of stem mustard in the southwestern China. In this study, different CMS-type specific mitochondrial markers were used to confirm that oxa CMS is distinct from the pol CMS, ogu CMS, nap CMS, hau CMS, tour CMS, Moricandia arvensis CMS, orf220-type CMS, etc., that have been previously reported in Brassica crops. Pollen grains of the oxa CMS line are sterile with a self-fertility rate of almost 0% and the sterility strain rate and sterility degree of oxa CMS is 100% due to a specific flower structure and flowering habit. Scanning electron microscopy revealed that most pollen grains in mature anthers of the oxa CMS line are empty, flat and deflated. Semi-thin section further showed that the abortive stage of anther development in oxa CMS is initiated at the late uninucleate stage. Abnormally vacuolated microspores caused male sterility in the oxa CMS line. This cytological study combined with marker-assisted selection showed that oxa CMS is a novel CMS type in stem mustard (Brassica juncea). Interestingly, the abortive stage of oxa CMS is later than those in other CMS types reported in Brassica crops, and there is no negative effect on the oxa CMS line growth period. This study demonstrated that this novel oxa CMS has a unique flower structure with sterile pollen grains at the late uninucleate stage. Our results may help to uncover the mechanism of oxa CMS in Brassica juncea.

     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of endangered <Emphasis Type="Italic">Mammillaria</Emphasis> genus (Cactaceae) species and genetic stability assessment using SSR markers

In vitro propagation protocols were established for endangered species of cacti Mammillaria hernandezii, M. dixanthocentron, and M. lanata. In vitro-germinated seedlings were used as the explant source. Three explant types were evaluated as apical, basal, and lateral stem sections. Shoot multiplication was achieved using Murashige and Skoog (MS) medium supplemented with benzyladenine, kinetin, meta-topolin, and thidiazuron in equimolar concentrations (0.0, 0.4, 1.1, 2.2, 4.4, and 8.9 μM). Shoot regeneration was obtained primarily in the lateral stem section explants. In M. hernandezii, an average of 7.4 shoots was regenerated in MS medium with 2.2 μM meta-topolin. M. dixanthocentron and M. lanata averaged 16.7 and 17.9 shoots/explant, respectively, in MS medium supplemented with 1.1 μM meta-topolin. Rooting occurred in MS medium without growth regulators. Three in vitro culture cycles were performed to validate the propagation protocols and to verify genetic stability. Shoots were collected in each cycle and genomic DNA was extracted. Amplified microsatellites were used to compare each genotype with its respective donor plant. Polymorphic information content analysis showed low levels of intra-clonal polymorphisms—M. hernandezii 0.04 and M. dixanthocentron and M. lanata both 0.12. More than 95% of the plants were successfully acclimatized in the greenhouse. After 12 months, plants of M. hernandezii reached the flowering stage; M. dixanthocentron and M. lanata flowered at 24 mo.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation and agronomic performance of regenerated chili pepper (C<Emphasis Type="Italic">apsicum</Emphasis> spp.) plants from commercially important genotypes

The application of modern biotechnology for improvement of chili pepper productivity requires an efficient in vitro plant regeneration protocol. In this study, a reliable protocol was developed for the in vitro regeneration of four types of chili, Capsicum annuum var. annuum (Jalapeño and Serrano), C. annuum var. glabriusculum/aviculare (Piquin), and C. chinense (Habanero) by direct organogenesis using three different explants (cotyledon, hypocotyls, and embryo) and three induction media. All evaluated culture media promoted the formation of adventitious shoots. When embryos or hypocotyls were used as explants, morphologically normal adventitious shoots developed, while culturing cotyledons resulted in nonelongating rosette-shaped shoots. The highest in vitro regeneration efficiency (14.6 shoots per explant) was achieved when Habanero chili hypocotyls were grown on Murashige and Skoog medium containing 1.7 μM indole-3-acetic acid and 22.2 μM N⁶-benzyladenine. This regeneration rate is higher than that obtained in previous reports. Regenerated plants were ready to be transferred to the greenhouse 13 wk after the explant culture. An evaluation carried out under greenhouse conditions showed differences in agronomic performance between in vitro regenerated plants and plants developed from seeds with the magnitude of the differences depending on the genotype being studied.     

Genetic structure of the populations of <Emphasis Type="Italic">Dactylorhiza ochroleuca</Emphasis> and <Emphasis Type="Italic">D. incarnata</Emphasis> (Orchidaceae) in the area of their joint growth in Russia and Belarus

We carried out an allozyme analysis to investigate polymorphism and genetic structure of the populations of D. incarnata and D. ochroleuca in regions of their joint growth in Russia and Belarus. We found that D. ochroleuca individuals in the populations of the Urals and Siberia, which are distant fragments from the main range of the species, do not differ significantly from individuals within the main part of the area (Belarus) on the basis of the allelic composition of eight gene loci. We revealed that D. ochroleuca and D. incarnata are differentiated by different alleles of the GDH locus. Thus, we established a genetic marker suitable to distinguish these closely related taxa. In addition to the GDH locus, D. ochroleuca and D. incarnata in the places of their joint growth, differ in the allelic structure of the PGI and NADHD loci. D. incarnata from the Urals and Siberia were polymorphic for both loci, and individuals from Belarus were polymorphic for one locus (PGI). In contrast, all D. ochroleuca individuals growing in sympatric populations with polymorphic D. incarnata were homozygous for the same alleles. Thus, comparison of the genetic structure of D. ochroleuca and D. incarnata points to the existence of a genetic isolation and a functioning isolation mechanism even under conditions of their joint growth. We found that the GDH locus in D. incarnata is polymorphic only in populations which grow together with D. ochroleuca, with exception a few examples. Thus, we conclude that variability of the GDH locus in D. incarnata is associated with hybridization with D. ochroleuca.