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Molecular characterization and expression patterns of a B-type nuclear lamin during sea urchin embryogenesis.

Developmentally regulated, tissue-specific patterns of nuclear lamin expression occur during vertebrate embryogenesis, but little is known regarding lamin ontogeny during the early development of other phyla. cDNA clones encoding a lamin from the sea urchins Strongylocentrotus purpuratus and Lytechinus variegatus have been identified, and the full coding region from the former has been sequenced. The predicted amino acid sequence indicates that this echinoderm lamin is more closely related to vertebrate B-type lamins than to dipteran fly and nematode lamins--the only other invertebrate lamins sequenced to date. Monoclonal and polyclonal antibodies to sea urchin lamin demonstrate that nuclei of unfertilized eggs and embryos exhibit relatively faint immunoreactivity until the differentiation of primary mesenchymal cells, the nuclear envelopes of which become strongly and selectively labeled by anti-lamin antibodies. Northern blots reveal stage-specific fluctuations in a single 4-kb lamin message during early development and, together with immunoblotting data, suggest that the increase in mesenchymal cell nuclear envelope immunoreactivity is due to a quantitative increase in a single type of lamin. These observations demonstrate that, similar to vertebrates, cell differentiation in invertebrates can be accompanied by a change in lamin expression patterns.

Amino Acid Sequence↗

Phosphorylation and localization of replication protein A during oogenesis and early embryogenesis of Drosophila melanogaster.

The phosphorylation and localization of Drosophila melanogaster Replication Protein A (DRP-A) was examined during oogenesis and in single embryos during the syncytial nuclear divisions of embryogenesis. DRP-A from ovaries was separated by two-dimensional electrophoresis into multiple phosphorylated species that include a previously unresolved form of RP-A. These forms are developmentally regulated with a major phosphorylated form appearing at stage 11 of oogenesis and persisting into mature eggs. Actively cycling early embryos were examined to investigate DNA replication in the absence of repair synthesis due to perturbation by drugs or mutation. An oscillation of the two major forms of DPR-A was observed over multiple cell cycles. The phosphorylated form was most abundant at mitosis and the nonphosphorylated form at interphase. In contrast to other systems where a phosphorylated form of RP-A has been correlated with S phase, only the nonphosphorylated form of Drosophila RP-A is observed in early Drosophila embryos during DNA replication. Consistent with this role in DNA metabolism, DRP-A was localized to the nucleus. Subsequently at mitosis, DRP-A becomes delocalized. Strikingly, in ovaries a relatively large amount of DRP-A was observed during the early mitotic stages of oogenesis.

Animals↗

A single MEF2 site governs desmin transcription in both heart and skeletal muscle during mouse embryogenesis.

Desmin, the muscle-specific intermediate filament protein is one of the earliest known myogenic makers both in heart and in somites. We have previously shown that high levels of desmin expression in the skeletal cell line C2C12 are due to a distal enhancer, which contains a muscle-specific factor-(2MEF2) binding site, adjacent to an E box, the binding site of the myogenic Helix-Loop-Helix (mHLH) regulators. We have further shown that MEF2C, a myocyte restricted member of the MEF2 family and all four mHLH factors can bind to their corresponding sites and through a cooperation with a second proximal E box can transactivate the desmin promoter. To study the significance of these regulatory elements in vivo, we have generated transgenic mice with desmin-lacZ reporters, intact or mutated at the MEF2 and E box of the enhancer. We show that the cis-acting DNA sequences within the 1-kb 5' flanking region of the mouse desmin gene are sufficient to direct appropriate temporal transcription both in heart and in skeletal muscle during mouse embryogenesis. Mutation at the MEF2 site completely suppressed transcription of the linked lacZ transgene in both developing heart and somites of the embryos. Mutation of the E box only suppressed activation in skeletal muscle precursors (somites and limb buds) but not in cardiac muscle. These data demonstrate that the MEF2 site is indispensable for the desmin enhancer function both in heart and in skeletal muscle. In addition, MEF2 cooperation with the mHLH regulators is absolutely necessary for proper transcriptional activity during embryonic skeletal muscle development.

Animals↗

Jagged2: a serrate-like gene expressed during rat embryogenesis.

DSL (Delta, Serrate, Lag-2) ligands activate Notch signaling and thereby regulate the differentiation of many different cell types during development. We have isolated a novel Serrate-like gene, Jagged2, whose amino acid sequence and expression pattern during rat embryogenesis suggest that it functions as a ligand for Notch. In contrast to previously described DSL ligands for Notch, Jagged2 is not widely expressed in the developing central nervous system. However, Jagged2 and Notch1 are coexpressed in the apical ectodermal ridge (AER), suggesting a role for this ligand-receptor pair in limb development. Furthermore, unlike Jagged1, Jagged2 is coexpressed with Notch in the developing thymus, where it may induce Notch signaling to direct T-cell fate. Our data are consistent with the idea that the diversity of cell types regulated by Notch signaling is a consequence of activation of unique Notch isoforms by different DSL ligands.

Amino Acid Sequence↗

The expression patterns of guidance receptors, DCC and Neogenin, are spatially and temporally distinct throughout mouse embryogenesis.

To gain a better understanding of the role of DCC and Neogenin in neural and nonneural tissues during vertebrate development we have carried out in situ hybridization studies to determine their expression patterns throughout the mid to late stages of mouse embryogenesis. This analysis revealed striking contrasts in both the spatial and temporal expression patterns of these closely related molecules. While DCC mRNA expression was predominantly restricted to the developing central nervous system (CNS), Neogenin mRNA was detected in a broad spectrum of embryonic tissues. Outside the CNS, Neogenin expression was observed mainly in mesodermal derivatives such as organ primordia and cartilage condensations of many developing embryonic structures. Within the CNS, initiation of DCC expression correlated with the onset of neurogenesis and was maintained at high levels in all regions of the developing CNS actively undergoing neurogenesis. By E18.5, DCC expression was detected only in structures such as the olfactory bulb, the hippocampus, and the cerebellum, that are known to sustain active neurogenesis well into postnatal life. In contrast, Neogenin expression was weak in the early developing CNS but broadened and intensified as neurogenesis proceeded. In summary, these observations indicate that Neogenin is the predominant member of this subfamily in mesodermal tissues, while DCC and Neogenin may play complementary roles in the generation of the fully functional CNS.

Animals↗

Regional specification during embryogenesis in the inarticulate brachiopod Discinisca.

The process of embryogenesis is described for the inarticulate brachiopod Discinisca strigata of the family Discinidae. A fate map has been constructed for the early embryo. The animal half of the egg forms the dorsal ectoderm of the apical and mantle lobes. The vegetal half forms mesoderm and endoderm and is the site of gastrulation; it also forms the ectoderm of the ventral regions of the apical and mantle lobes of the larva. The plane of the first cleavage goes through the animal-vegetal axis of the egg along the future plane of bilateral symmetry of the larva. The timing of regional specification in these embryos was examined by isolating animal, vegetal, or lateral regions at different times from the 2-cell stage through gastrulation. Animal halves isolated at the 8-cell and blastula stages formed an epithelial vesicle and did not gastrulate. When these halves were isolated from blastulae they formed the cell types typical of apical and mantle lobes. Vegetal halves isolated at all stages gastrulated and formed a more or less normal larva; the only defect these larvae had was the lack of an apical tuft, which normally forms from cells at the animal pole of the embryo. When lateral isolates were created at all developmental stages, these halves gastrulated. Cuts which separated presumptive anterior and posterior regions generated isolates at the 4-cell and blastula stages that formed essentially normal larvae; however, at the midgastrula stage these halves formed primarily anterior or posterior structures indicating that regional specification had taken place along the anterior-posterior axis. The plane of the first cleavage, which predicts the plane of bilateral symmetry, can be shifted by either changing the cleavage pattern that generates the bilateral 16-cell blastomere configuration or by isolating embryo halves prior to, or during, the 16-cell stage. These results indicate that while the plane of the first cleavage predicts the axis of bilateral symmetry, the axis is not established until the fourth cleavage. The development of Discinisca is compared to development in the inarticulate brachiopod Glottidia of the family Lingulidae and to Phoronis in the phylum Phoronida.

Animals↗

Multiple roles for activin-like kinase-2 signaling during mouse embryogenesis.

The members of the transforming growth factor-beta (TGF-beta) superfamily are secreted proteins that interact with cell-surface receptors to elicit signals that regulate a variety of biological processes during vertebrate embryogenesis. Alk2, also known as ActRIA, Tsk7L, and SKR1, encodes a type I TGF-beta family receptor for activins and BMP-7. Initially, Alk2 transcripts are detected in the visceral endoderm of gastrula stage mouse embryos, suggesting a signaling role in extraembryonic tissues during development. To study the role of Alk2 during mammalian development, Alk2 mutant mice were generated. After embryonic day 9.5 (E9.5), no homozygous mutants were recovered from heterozygote matings. Homozygous mutants with morphological defects were first detected at E7.0 and were smaller than controls. Morphological and molecular examination demonstrated that Alk2 mutant embryos formed a primitive streak, although abnormally thickened, and were arrested in their development around the late streak stage. These gastrulation defects were rescued in chimeric embryos generated by injection of Alk2 mutant embryonic stem (ES) cells into wild-type blastocysts. This rescue of gastrulation defects was also observed in chimeric embryos generated by aggregation of Alk2 homozygous mutant ES cells with tetraploid wild-type embryos. However, at E9.5, these embryos that were completely ES-derived also had defects. In contrast, chimeric embryos generated by injection of wild-type ES cells into Alk2 mutant blastocysts did not show rescue of the gastrulation defects. These results suggest that signaling through this type I receptor is essential in extraembryonic tissues at the time of gastrulation for normal mesoderm formation and also suggest that subsequent Alk2 signaling is essential for normal development after gastrulation.

Activin Receptors, Type I↗

Spatial and temporal patterns of cell division during early Xenopus embryogenesis.

We describe the spatial and temporal patterns of cell division in the early Xenopus embryo, concentrating on the period between the midblastula transition and the early tailbud stage. Mitotic cells were identified using an antibody recognising phosphorylated histone H3. At least four observations are of interest. First, axial mesodermal cells, including prospective notochord, stop dividing after involution and may not divide thereafter. Second, cell division is more pronounced in the neural plate than in nonneural ectoderm, and the pattern of cell division becomes further refined as neurogenesis proceeds. Third, cells in the cement gland cease proliferation completely as they begin to accumulate pigment. Finally, the precursors of peripheral sensory organs such as the ear and olfactory placode undergo active cell proliferation when they arise from the sensorial layer of the ectoderm. These observations and others should provide a platform to study the relationship between the regulation of developmental processes and the cell cycle during Xenopus embryogenesis.

Animals↗

Zygotic expression of the caudal homolog pal-1 is required for posterior patterning in Caenorhabditis elegans embryogenesis.

Previous work has shown that the Caenorhabditis elegans gene pal-1, a homolog of Drosophila caudal, is required maternally for blastomere specification in the early embryo and postembryonically for tail development in males. We show here that embryonic (zygotic) transcription of pal-1 is also required for posterior patterning during later embryogenesis. Embryos homozygous for strong loss-of-function mutations arrest as nonviable L1 larvae with gross posterior defects. PAL-1 protein produced from zygotic transcripts is expressed dynamically during gastrulation and morphogenesis in specific cells of all major lineages except the germ line. Most expressing cells are undergoing cell movements or forming midline structures or both. Mutant embryos exhibit defects involving most of the expressing cells. Aberrant early cell positions are observed in posterior hypodermis, both in the C-lineage cells that express pal-1 and in the neighboring hypodermal seam cell precursors, which do not, as well as in posterior muscle derived from the C and D lineages. Defects in late gastrulation, ventral hypodermal enclosure, and formation of the rectum result from failures of cell movements of ABp and MS descendants. Limited mosaic analysis supports the view that most of the required pal-1 functions are cell autonomous.

Animals↗

Multiple nodal-related genes act coordinately in Xenopus embryogenesis.

Four nodal-related genes (Xnr1-4) have been isolated in Xenopus to date, and we recently further identified two more, Xnr5 and Xnr6. In the present functional study, we constructed cleavage mutants of Xnr5 (cmXnr5) and Xnr6 (cmXnr6) which were expected to act in a dominant-negative manner. Both cmXnr5 and cmXnr6 inhibited the activities of Xnr5 and Xnr6 in co-overexpression experiments. cmXnr5 also inhibited the activity of Xnr2, Xnr4, Xnr6, derrière, and BVg1, but did not inhibit the activity of Xnr1 or activin. Misexpression of cmXnr5 led to a severe delay in initiation of gastrulation and phenotypic changes, including defects in anterior structures, which were very similar to those seen in maternal VegT-depleted embryos. Further, although the expression of Xnr1, Xnr2, and Xnr4 was not delayed in these embryos, it was markedly reduced. Injection of cmXnr5 had no notable effect on expression of Xnr3, Xnr6, derrière, or siamois. Several mesodermal and endodermal markers also showed delayed and decreased expression during gastrulation in cmXnr5-injected embryos. These results suggest that, in early Xenopus embryogenesis, nodal-related genes may heterodimerize with other TGF-beta ligands, and further that one nodal-related gene alone is insufficient for mesendoderm formation, which may require the cooperative interaction of multiple nodal-related genes.

Amino Acid Sequence↗

The immunolocalization of nuclear antigens during the pollen developmental program and the induction of pollen embryogenesis.

The immunolocalization of nuclear antigens, combined with cytochemical procedures as well as in situ hybridization and recent in situ molecular methods, has been applied at different steps of pollen development to characterize the functional organization of the nucleus during the formation of the male gametophyte in an agronomically interesting plant, Capsicum annuum L. Pollen embryogenesis has been induced in pepper and the first stages of the process have been studied at the cellular level. Low temperature processing methods including cryosections and Lowicryl sections were very convenient for performing the various in situ techniques used in the pollen grains. Different molecular probes for localizing DNA, RNA, snRNPs, specific nucleolar proteins, various rRNA species, and DNA/RNA hybrids provided positive results in the pollen nuclei. The data obtained, and the changes observed in the organization of the nuclear compartments during pollen development, are related to the variations in gene activity undergone by the male gametophyte. The methodology used is proposed as a very convenient approach to localize molecules and events involved in the nuclear function in both gametophytic and sporophytic pollen development.

Antigens↗

The metabolism of biogenic monoamines during embryogenesis and metamorphosis in two anuran species.

This study investigated the pathways to many monoamines and their metabolites in the central nervous system of the frog Rana nigromaculata and the toad Bufo bufo japonicus during embryonic development and metamorphosis. Metabolites were analyzed by three-dimensional HPLC. The two species provided evidence of similar pathways, with slightly different timetables for the development of their monoamine systems. During embryonic development, the main metabolic pathways in entire embryos were tyrosine (TYR)-->[3,4-dihydroxyphenylalanine in Bufo]-->3-hydroxytyramine-->norepinephrine or epinine (EPIN)-->epinephrine, TYR-->tyramine--> (octopamine in Rana) and TYR-->3-O-methyldopa for catecholamines, and tryptophan-->kynurenine and 5-hydroxytryptamine (5-HT)-->[5-hydroxyindoleacetic acid and N-methyl-5-hydroxytryptamine (N-MET) in Bufo]. The monoamine system in the brain was similar during metamorphosis to that during embryogenesis with a few exceptions. The most striking change was the development of the bufotenine (5-hydroxy-N, N-dimethyltryptamine) pathway from 5-HT via N-MET. EPIN and norepinephrine in Rana and octopamine in both species disappeared during metamorphosis. These results are discussed in relation to the roles of the various pathways in development.

Animals↗

Expression of cytochrome P450 cholesterol side chain cleavage and 3beta-hydroxysteroid dehydrogenase during embryogenesis in chicken adrenal glands and gonads.

Expression of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNAs was examined in chicken embryonic adrenal glands and gonads between days 4 and 12 of incubation. In situ hybridization analysis showed that 3beta-HSD mRNA appeared on day 5 of incubation in the adrenal glands and on day 6 in the gonads, while P450scc mRNA was expressed on day 7 in both the adrenal glands and the gonads. Cells expressing both enzyme mRNAs were distributed in the steroidogenic tissues of the adrenal glands and in the medullary cords of the gonads. From days 9 to 11 of incubation, P450scc mRNA expression was not found in the majority of both the adrenal glands and the gonads, but was detected again in both on day 12, although 3beta-HSD mRNA was constitutively expressed during this period. Changes in the expression pattern of P450scc mRNA are paralleled by changes in the plasma corticosterone level reported previously. Therefore, it is suggested that P450scc is essential to embryogenesis.

3-Hydroxysteroid Dehydrogenases↗

Identification of target genes regulated by PAX3 and PAX3-FKHR in embryogenesis and alveolar rhabdomyosarcoma.

PAX3 is a transcription factor important for neural, muscle, and facial development in vertebrates. To identify genes regulated by PAX3, we used a cyclic amplification and selection of targets (CASTing) strategy to isolate cis-regulatory elements bound by PAX3. CASTing libraries were constructed with mouse DNA fragments bound by mouse PAX3, and human genomic DNA fragments bound by human PAX3 and the fusion protein PAX3-FKHR. Approximately 1000 clones were sequenced from each of these three libraries. Numerous putative targets of PAX3 and PAX3-FKHR were identified and six genes, Itm2A, Fath, FLT1, TGFA, BVES, and EN2, were examined closely. The genomic DNA fragments near these genes contain PAX3 binding sites and confer PAX3-dependent regulation. The expression levels of these genes correlate with the PAX3 expression levels in mouse embryos or with PAX3-FKHR expression levels in rhabdomyosarcoma cell lines, and indicate they may be part of the PAX3 regulatory circuitry during embryogenesis and tumor formation.

Animals↗

Pattern formation in plant embryogenesis: a reassessment.

Pattern formation in embryogenesis generates the basic body plan of flowering plants by establishing the diversity of position-dependent cell fates characteristic of the seedling. The concept of pattern formation, which was originally based on the analysis of Arabidopsis pattern mutants, is re-examined in the light of recent data that address the origin of the axis of polarity, the origin of the primary meristems as elements of the apical-basal pattern and the formation of the radial pattern of tissue layers. The available evidence from genetic, molecular and experimental approaches supports the notion that embryonic pattern formation occurs in steps involving communication between clonally unrelated cells.

Body Patterning↗

Production of Agrobacterium-mediated transgenic fertile plants by direct somatic embryogenesis from immature zygotic embryos of Datura innoxia.

This work describes a new method to obtain transgenic somatic embryos from Agrobacterium-infected immature zygotic embryos of Datura innoxia. It has several advantages over previous transformation methods such as the absence of a callus phase, an average transformation rate of 76% and a high regeneration frequency. Critical steps for optimal transformation were the embryo stage and a short preculture treatment. The marker gene beta-glucuronidase and light microscopy were used to identify the competent embryogenic cells which, after transformation, passed through the classical stages of embryo development. The transgenes were transmitted to the progeny in a Mendelian fashion. The plants regenerated via direct somatic embryogenesis were cytologically and morphologically uniform. We also observed that: (1) wounding or wound-induced divisions were not required for zygotic embryo transformation; (2) epidermal cells were competent for both transformation and regeneration; and (3) competency for Agrobacterium infection was developmental stage-specific. This new method should facilitate the development of new strategies to routinely transform recalcitrant plant species.

Datura stramonium↗

PCR-generated cDNA library of transition-stage maize embryos: cloning and expression of calmodulin genes during early embryogenesis.

One hundred maize zygotic embryos microdissected at the transition stage were used to construct a cDNA library after non-selective PCR (NS-PCR) amplification of whole cDNA populations. The library contains 2.3 x 10(5) recombinants and two different calmodulin cDNAs were cloned using a heterologous probe from petunia. Calmodulin expression was confirmed throughout maize embryogenesis at the mRNA, amplified cDNA and protein levels. Sequence analysis suggests a maize origin for both clones and negligible nucleotide changes linked to PCR. This library is the first described for early plant embryos and represents a breakthrough to isolate genes involved in embryo differentiation.

Amino Acid Sequence↗

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis.

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter-beta-glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.

2S Albumins, Plant↗