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The retinoblastoma gene family is differentially expressed during embryogenesis.

We report differential expression of the RB1 tumor suppressor gene and the homologous genes p107 and p130 during embryogenesis. Abundant RB1 transcripts were detected during neurogenesis, hematopoiesis, myogenesis, lens development and in the ganglion cell layer of the embryonic retina, prior to and during differentiation. The expression pattern of RB1 mirrored the defects in RB1 mutant mice (RB-/-). In the heart, lung, kidney and intestine, p107, but not RB1, was expressed. In the liver and the central nervous system p107 and RB1 were co-expressed, consistent with the accelerated cell death observed in RB-/-; p107-/- double knock-out mice. In the central nervous system, p107 expression was restricted to proliferating cells surrounding the ventricles, while RB1 was expressed in areas of both proliferating and differentiating cells. In contrast to RB1 and p107, expression of p130 was low throughout embryogenesis. In situ hybridization and Western blot analyses showed that the expression of p107 and p130 was not markedly altered in RB-/- embryos compared to control littermates. Our results suggest that members of RB1 gene family have distinct, but overlapping roles in embryogenesis, with p107 and RB1 possibly having redundant functions in the central nervous system and liver.

Animals↗

Spaceflight reduces somatic embryogenesis in orchardgrass (Poaceae).

Somatic embryos initiate and develop from single mesophyll cells in in vitro cultured leaf segments of orchard-grass (Dactylis glomerata L.). Segments were plated at time periods ranging from 21 to 0.9 d (21 h) prior to launch on an 11 d spaceflight (STS-64). Using a paired t-test, there was no significant difference in embryogenesis from preplating periods of 14 d and 21 d. However, embryogenesis was reduced by 70% in segments plated 21 h before launch and this treatment was significant at P=0.0001. The initial cell divisions leading to embryo formation would be taking place during flight in this treatment. A higher ratio of anticlinal:periclinal first cell divisions observed in the flight compared to the control tissue suggests that microgravity affects axis determination and embryo polarity at a very early stage. A similar reduction in zygotic embryogenesis would reduce seed formation and have important implications for long-term space flight or colonization where seeds would be needed either for direct consumption or to grow another generation of plants.

Chromosome Aberrations↗

Expression and cellular localization of rab28 mRNA and Rab28 protein during maize embryogenesis.

The maize abscisic acid (ABA) responsive gene rab28, has been shown to be ABA-inducible in embryos and vegetative tissues. A polyclonal antiserum was raised against Rab28 protein. Using immunoblotting and immunoprecipitation, the antiserum specifically recognized a protein of about 30 kDa and pl 6 which is in close agreement with the molecular weight and pl predicted by the deduced amino acid sequence. The rab28 gene product accumulated during late embryogenesis. In vegetative tissues, dehydration stress induced rab28 gene expression both in the light and in the dark. The spatial and temporal pattern of rab28 mRNA expression during embryogenesis was investigated by in situ hybridization using digoxigenin-labelled rab28 probes, and the immunochemical localization of Rab28 protein using anti-Rab28 antibodies. Expression of rab28 mRNA is restricted to provascular tissues in young embryos, and at later stages of development the most prevalent accumulation occurred in meristem and in the vascular elements of the plumule, root and scutellum. Using immunoelectron microscopy the Rab28 protein has been located in the nucleolus of different cell types. In light of these results the stress regulation of rab28 and a likely role for this protein during late embryogenesis are discussed.

Antibody Specificity↗

Position dependent expression of GL2-type homeobox gene, Roc1: significance for protoderm differentiation and radial pattern formation in early rice embryogenesis.

In early plant embryogenesis, the determination of cell fate in the protodermal cell layer is considered to be the earliest event in radial pattern formation. To elucidate the mechanisms of epidermal cell fate determination and radial pattern formation in early rice embryogenesis, we have isolated a GL2-type homeobox gene Roc1 (Rice outermost cell-specific gene1), which is specifically expressed in the protoderm (epidermis). In early rice embryogenesis, cell division occurs randomly and the morphologically distinct layer structure of the protoderm cannot be observed until the embryo reaches more than 100 microm in length. Nonetheless, in situ hybridization analyses revealed that specific expression of Roc1 in the outermost cells is established shortly after fertilization, much earlier than protoderm differentiation. In the regeneration process from callus, the Roc1 gene is also expressed in the outermost cells of callus in advance of tissue and organ differentiation, and occurs independently of whether the cells will differentiate into epidermis in the future or not. Furthermore, this cell-specific Roc1 expression could be induced flexibly in the newly produced outermost cells when we cut the callus. These findings suggest that the expression of Roc1 in the outermost cells may be dependent on the positional information of cells in the embryo or callus prior to the cell fate determination of the protoderm (epidermis). Furthermore, the Roc1 expression is downregulated in the inner cells of ligule, which have previously been determined as protodermal cells, also suggesting that the Roc1 expression is position dependent and that this position dependent Roc1 expression is important also in post-embryonic protoderm (epidermis) differentiation.

Arabidopsis Proteins↗

Module-specific regulation of the beta-phaseolin promoter during embryogenesis.

The phas promoter displays stringent spatial regulation, being very highly expressed during embryogenesis and completely silent during all phases of vegetative development in bean, Phaseolus vulgaris. This pattern is maintained in transgenic tobacco and, as shown here, Arabidopsis. Dimethyl sulphate in vivo footprinting analyses revealed that over 20 cis-elements within the proximal 295 bp of the phas promoter are protected by factor binding in seed tissues whereas none are bound in leaves. The hypothesis that this complex profile represents a summation of several module (cotyledon, hypocotyl, and radicle)-specific factor-DNA interactions has been explored by the incorporation of site-directed substitution mutations into 10 locations within the -295phas promoter. Only 2.6% of -295phas promoter activity remained after mutation of the G-box; the CCAAAT box, the E-box and the RY elements were also found to mediate high levels of expression in embryos. Whereas the CACA element has dual positive and negative regulatory roles, the vicilin box was identified as a strong negative regulatory element. The proximal (-70 to -64) RY motif was found to bestow expression in the hypocotyl while all the RY elements contribute to expression in cotyledons but not to vascular tissue expression during embryogenesis. RY elements at positions -277 to -271, -260 to -254, and -237 to -231 were found to orchestrate radicle-specific repression. The G-box appears to be the functional abscisic acid responsive element and the E-site may be a coupling element. The results substantiate the concept that autarkical cis-element functions generate modular patterning during embryogenesis. They also reflect the existence of both redundancy and hierarchy in cis-element interactions. Importantly, the virtually identical expression patterns observed for the two distantly related plants studied argue strongly for the generality of function for the observed factor-element interactions.

Arabidopsis↗

Glucocorticoid receptor gene expression during rat embryogenesis. An in situ hybridization study.

Glucocorticoids play an important role in embryonic development. The existence of sufficient amounts of their receptors during rodent embryogenesis has proved to be an absolute necessity for the physiological growth of the animal. We have analyzed the pattern of glucocorticoid receptor gene expression in the rat embryo through embryonic days 12 to 17, by using in situ hybridization histochemistry. Glucocorticoid receptor mRNA is present in the rat liver on embryonic day (E) 12, and by E13 the signal can also be detected in several other tissues, such as the lung, the heart, the mesonephros, the sclerotomes, the thymus and Rathke's pouch. Glucocorticoid receptor gene expression was quite ubiquitous in tissue derivatives of all three germ layers and appeared to vary in intensity within the same tissue during embryogenesis. These variations in the level of receptor gene expression paralleled the developmental stage of each tissue: Intense labelling was detected just prior to the final differentiation step of a structure. Upon differentiation, cell populations highly expressing glucocorticoid receptor gene in the previous stage were found to have reduced amounts of the receptor mRNA. Our results support a morphogenetic role for glucocorticoids during embryogenesis.

Animals↗

Embigin/basigin subgroup of the immunoglobulin superfamily: different modes of expression during mouse embryogenesis and correlated expression with carbohydrate antigenic markers.

Embigin and basigin are highly glycosylated transmembrane glycoproteins with two immunoglobulin domains and form a subgroup in the immunoglobulin superfamily. Previous studies have demonstrated the functional significance of these molecules. In the present study, in situ hybridization analysis of their expression was performed during mouse embryogenesis. Embigin was strongly expressed in the endoderm during early postimplantation embryogenesis, and in the somite stage in the gut and visceral endoderm. Embryonic ectoderm and its derivative tissues weakly to moderately expressed this molecule. From day 10 to 15 of gestation, no embigin signal was detected. Basigin was more broadly expressed. During the organogenesis period, basigin was expressed in various epithelial tissues, brain ventricles, the spinal cord and dorsal root ganglion. The modes of expression of these two proteins throughout the egg cylinder stage correlated with the expression of the carbohydrate markers that they carry; embigin with Dolichos biflorus agglutinin binding sites and basigin with LeX antigen and more closely with fucosyltransferase IV, which forms the antigenic epitope. These findings imply that proteins with specific carbohydrate epitopes play roles in early postimpantation embryogenesis.

Animals↗

Telomere length is reset during early mammalian embryogenesis.

The enzyme telomerase is active in germ cells and early embryonic development and is crucial for the maintenance of telomere length. Whereas the different length of telomeres in germ cells and somatic cells is well documented, information on telomere length regulation during embryogenesis is lacking. In this study, we demonstrate a telomere elongation program at the transition from morula to blastocyst in mice and cattle that establishes a specific telomere length set point during embryogenesis. We show that this process restores telomeres in cloned embryos derived from fibroblasts, regardless of the telomere length of donor nuclei, and that telomere elongation at this stage of embryogenesis is telomerase-dependent because it is abrogated in telomerase-deficient mice. These data demonstrate that early mammalian embryos have a telomerase-dependent genetic program that elongates telomeres to a defined length, possibly required to ensure sufficient telomere reserves for species integrity.

Animals↗

Evidence that insect embryogenesis is regulated by ecdysteroids released from yolk proteins.

That the yolk proteins (YPs), or vitellins, stored in the oocytes of insects are a nutritional store for subsequent embryogenesis has long been assumed. Exhaustive data base searching programs revealed highly significant sequence similarity between the three YPs of Drosophila melanogaster and part of the triacylglycerol lipase of the domestic pig. Based upon time of degradation of YPs during embryogenesis, existence of maternally stored ecdysteroid conjugates in embryos, location of these conjugates in locust embryos, and the fact that free active ecdysteroid hormones are released at a specific time in embryogenesis to trigger cuticle deposition, we postulate that the similarity reflects a common property of Drosophila YPs--the ability to bind the fatty acid ecdysteroid conjugates. Our finding of conjugated ecdysteroids tightly bound to purified Drosophila YP supports this prediction.

Amino Acid Sequence↗

A rice homeobox gene, OSH1, is expressed before organ differentiation in a specific region during early embryogenesis.

Homeobox genes encode a large family of homeodomain proteins that play a key role in the pattern formation of animal embryos. By analogy, homeobox genes in plants are thought to mediate important processes in their embryogenesis, but there is very little evidence to support this notion. Here we described the temporal and spatial expression patterns of a rice homeobox gene, OSH1, during rice embryogenesis. In situ hybridization analysis revealed that in the wild-type embryo, OSH1 was first expressed at the globular stage, much earlier than organogenesis started, in a ventral region where shoot apical meristem and epiblast would later develop. This localized expression of OSH1 indicates that the cellular differentiation has already occurred at this stage. At later stages after organogenesis had initiated, OSH1 expression was observed in shoot apical meristem [except in the L1 (tunica) layer], epiblast, radicle, and their intervening tissues in descending strength of expression level with embryonic maturation. We also performed in situ hybridization analysis with a rice organless embryo mutant, orl1, that develops no embryonic organs. In the orl1 embryo, the expression pattern of OSH1 was the same as that in the wild-type embryo in spite of the lack of embryonic organs. This shows that OSH1 is not directly associated with organ differentiation, but may be related to a regulatory process before or independent of the organ determination. The results described here strongly suggest that, like animal homeobox genes, OSH1 plays an important role in regionalization of cell identity during early embryogenesis.

Cell Differentiation↗

Detection of targeted GFP-Hox gene fusions during mouse embryogenesis.

The ability to use a vital cell marker to study mouse embryogenesis will open new avenues of experimental research. Recently, the use of transgenic mice, containing multiple copies of the jellyfish gene encoding the green fluorescent protein (GFP), has begun to realize this potential. Here, we show that the fluorescent signals produced by single-copy, targeted GFP in-frame fusions with two different murine Hox genes, Hoxa1 and Hoxc13, are readily detectable by using confocal microscopy. Since Hoxa1 is expressed early and Hoxc13 is expressed late in mouse embryogenesis, this study shows that single-copy GFP gene fusions can be used through most of mouse embryogenesis. Previously, targeted lacZ gene fusions have been very useful for analyzing mouse mutants. Use of GFP gene fusions extends the benefits of targeted lacZ gene fusions by providing the additional utility of a vital marker. Our analysis of the Hoxc13(GFPneo) embryos reveals GFP expression in each of the sites expected from analysis of Hoxc13(lacZneo) embryos. Similarly, Hoxa1(GFPneo) expression was detected in all of the sites predicted from RNA in situ analysis. GFP expression in the foregut pocket of Hoxa1(GFPneo) embryos suggests a role for Hoxa1 in foregut-mediated differentiation of the cardiogenic mesoderm.

Aging↗

Expression of the smooth muscle cell calponin gene marks the early cardiac and smooth muscle cell lineages during mouse embryogenesis.

Although several genes are considered markers for vascular smooth muscle cell (SMC) differentiation, few have been rigorously tested for SMC specificity in mammals, particularly during development where considerable overlap exists between different muscle gene programs. Here we describe the temporospatial expression pattern of the SMC calponin gene (formerly h1 or basic calponin) during mouse embryogenesis and in adult mouse tissues and cell lines. Whereas SMC calponin mRNA expression is restricted exclusively to SMCs in adult tissues, during early embryogenesis, SMC calponin transcripts are expressed throughout the developing cardiac tube as well as in differentiating SMCs. Transcription of the SMC calponin gene initiates at two closely juxtaposed sites in the absence of a consensus TATAA or initiator element. Transient transfection assays in cultured SMC demonstrated that high level SMC calponin promoter activity required no more than 549 nucleotides of 5 sequence. In contrast to the strict cell type-specificity of SMC calponin mRNA expression, the SMC calponin promoter showed activity in several cell lines that do not express the endogenous SMC calponin gene. These results demonstrate that SMC calponin responds to cardiac and smooth muscle gene regulatory programs and suggest that the cardiac and smooth muscle cell lineages may share a common gene regulatory program early in embryogenesis, which diverges as the heart matures. The finding that the isolated SMC calponin promoter is active in a wider range of cells than the endogenous SMC calponin gene also suggests that long-range repression or higher order regulatory mechanism(s) are involved in cell-specific regulation of SMC calponin expression.

Animals↗

Cathepsin L is essential for embryogenesis and development of Caenorhabditis elegans.

Cysteine proteases play critical biological roles in both intracellular and extracellular processes. We characterized Ce-cpl-1, a Caenorhabditis elegans cathepsin L-like cysteine protease. RNA interference with Ce-cpl-1 activity resulted in embryonic lethality and a transient delayed growth of larvae to egg producing adults, suggesting an essential role for cpl-1 during embryogenesis, and most likely during post-embryonic development. Cpl-1 gene (Ce-cpl-1:lacZ) is widely expressed in the intestine and hypodermal cells of transgenic worms, while the fusion protein (Ce-CPL-1::GFP) was expressed in the hypodermis, pharynx, and gonad. The CPL-1 native protein accumulates in early to late stage embryos and becomes highly concentrated in gut cells during late embryonic development. CPL-1 is also present near the periphery of the eggshell as well as in the cuticle of larval stages suggesting that it may function not only in embryogenesis but also in further development of the worm. Although the precise role of Ce-CPL-1 during embryogenesis is not yet clear it could be involved in the processing of nutrients responsible for synthesis and/or in the degradation of eggshell. Moreover, an increase in the cpl-1 mRNA is seen in the intermolt period approximately 4 h prior to each molt. During this process Ce-CPL-1 may act as a proteolytic enzyme in the processing/degradation of cuticular or other proteins. Similar localization of a related cathepsin L in the filarial nematode Onchocerca volvulus, eggshell and cuticle, suggests that some of the Ce-CPL-1 function during development may be conserved in other parasitic nematodes.

Amino Acid Sequence↗

Characterization of the Xenopus galectin family. Three structurally different types as in mammals and regulated expression during embryogenesis.

We have isolated six novel galectin cDNAs from a Xenopus laevis kidney cDNA library. The newly identified X. laevis galectins (xgalectins) comprise one proto type (xgalectin-Vb), one chimera type (xgalectin-VIIa), and four tandem repeat types (xgalectin-IIb, -IIIb, -VIa, and -VIIIa). Thus, together with those mentioned in our previous work (Shoji, H., Nishi, N., Hirashima, M., and Nakamura, T. (2002) Glycobiology 12, 163-172), the 12 xgalectins are classified into three types based on their domain structures, as in mammals. The xgalectins whose counterparts in other species have not been identified (xgalectin-IVa, -Vb, and -VIa) were confirmed to possess lactose-binding activity by expression of their recombinant forms. This shows that they truly function as animal lectins. The protein purification study revealed that the major xgalectins in kidney are xgalectin-Ib, -IIa, -IIb, -IIIa, and -VIIa. The mRNAs of xgalectin-IIb, -IIIb, -Vb, and -VIa were localized to specific adult tissues, whereas those of xgalectin-VIIa and -VIIIa were broadly distributed. The temporal expression patterns of the mRNAs of the 12 xgalectins during embryogenesis were analyzed and categorized into three groups: 1) mRNA observed to exist throughout embryogenesis, i.e. maternal mRNA also exists (xgalectin-Ia, -IIa, -IIIa, -IIIb, -Va, -VIIa, and -VIIIa); 2) mRNA observed from the gastrula stage (xgalectin-VIa); and 3) mRNA observed from the tail bud or the tadpole stage (xgalectin-Ib, -IIb, -IVa, and -Vb). The mRNA of the most abundant xgalectin in embryos, xgalectin-VIIa, was localized to the surface layer of embryos, the epidermis, the cement gland, and various placodes. Xgalectin-VIIa protein was also observed to exist throughout embryogenesis by Western blot analysis with specific antiserum. These results show that the expression of each member is spatiotemporally regulated from eggs to adulthood, suggesting that galectins play multiple roles not only in adults, but also in development.

Amino Acid Sequence↗

Yaf2 inhibits caspase 8-mediated apoptosis and regulates cell survival during zebrafish embryogenesis.

Rybp (DEDAF) is a member of the Rybp/Yaf2 protein family and has been shown to encode pro-apoptotic functions and to be essential for mouse embryogenesis. The related Yaf2 protein has not been studied extensively at the cellular or organismal levels. Here we describe zebrafish yaf2 (zyaf2) and show that it is widely expressed during early embryogenesis, with subsequent enrichment of transcripts in the anterior head region. Depletion of zYaf2 during embryogenesis using specific morpholinos activates a wide-spread program of apoptosis and causes developmental arrest before the one somite stage. Partial depletion of Yaf2, achieved by injecting lower dosages of morpholino, circumvents the early arrest but leads to CNS degeneration associated with excessive apoptosis. These phenotypes can be rescued by co-injection of human YAF2 mRNA with the morpholinos or by treatment with a pan-caspase inhibitor or a caspase 8-specific inhibitor. Finally, the observed activation of caspase 8 in the morphants is in accord with the ability of Yaf2 to inhibit caspase 8-mediated apoptosis in cultured cells. Our findings implicate Yaf2 as a survival factor during early zebrafish development and organogenesis. This may suggest that Yaf2 and Rybp can encode opposing functions in the regulation of apoptosis.

Amino Acid Sequence↗

Somatic embryogenesis and plant regeneration from cotyledon explants of a timber-yielding leguminous tree, Dalbergia sissoo Roxb.

Efficient plant regeneration through somatic embryogenesis was achieved from callus cultures derived from semi-mature cotyledon explants of Dalbergia sissoo Roxb., a timber-yielding leguminous tree. Somatic embryos developed over the surface of embryogenic callus and occasionally, directly from cotyledon explants without intervening callus phase. Callus cultures were initiated from cotyledon pieces of D. sissoo on Murashige and Skoog (1962) medium supplemented with 4.52, 9.04, 13.57, and 18.09 mumol/L 2,4-dichlorophenoxyacetic acid and 0.46 mumol/L Kinetin. Maximum percentage response for callus formation was 89% on MS medium supplemented with 9.04 mumol/L 2,4-D' and 0.46 mumol/L Kn. Somatic embryogenesis was achieved after transfer of embryogenic callus clumps to 1/2-MS medium without plant growth regulators (1/2-MSO). Average numbers of somatic embryos per callus clump was 26.5 on 1/2-MSO medium after 15 weeks of culture. Addition of 0.68 mmol/L L-glutamine to 1/2-MSO medium enhanced somatic embryogenesis frequency from 55% to 66% and the number of somatic embryos per callus clump from 26.5 to 31.1. Histological studies were carried out to observe various developmental stages of somatic embryos. About 50% of somatic embryos converted into plantlets on 1/2-MSO medium containing 2% sucrose, after 20 days of culture. Transfer of somatic embryos to 1/29-MSO medium containing 10% sucrose for 15 days prior to transfer on 1/2-MS medium with 2% sucrose enhanced the conversion of somatic embryos into plantlets from 50 to 75%. The plantlets with shoots and roots were transferred to 1/2 and 1/4-liquid MS medium, each for 10 days, and then to plastic pots containing autoclaved peat moss and compost mixture (1:1). 70% of the plantiets survived after 10 weeks of transfer to pots. 120 regenerated plantlets out of 150 were successfully acclimatised. After successful acclimatisation, plants were transferred to earthen pots.

Fabaceae↗

Hemoglobin promotes somatic embryogenesis in peanut cultures.

Critical parameters influencing somatic embryogenesis include growth regulators and oxygen supply. Consequently, the present investigation has focused on optimization of a somatic embryogenic system for peanut (Arachis hypogaea L.) through media supplementation with the auxin, picloram. The latter at 30 mg L(-1) was optimal for inducing regeneration of somatic embryos from cultured explants of zygotic embryos. In contrast, somatic embryogenesis did not occur in the absence of this growth regulator. An assessment has also been made of the beneficial effect on somatic embryogenesis and plant regeneration of the commercial hemoglobin (Hb) solution, Erythrogen. Hemoglobin at 1:50 and 1:100 (v:v) stimulated increases in mean fresh weight (up to a maximum of 57% over control), mean number of explants producing somatic embryos (15%) and mean number of somatic embryos per explant (29%).

Arachis↗

Caenorhabditis elegans decapping proteins: localization and functional analysis of Dcp1, Dcp2, and DcpS during embryogenesis.

Though posttranscriptional regulation is important for early embryogenesis, little is understood regarding control of mRNA decay during development. Previous work defined two major pathways by which normal transcripts are degraded in eukaryotes. However it is not known which pathways are key in mRNA decay during early patterning or whether developmental transcripts are turned over via specific pathways. Here we show that Caenorhabditis elegans Dcp2 is localized to distinct foci during embryogenesis, reminiscent of P-bodies, the sites of mRNA degradation in yeast and mammals. However the decapping enzyme of the 3' to 5' transcript decay system (DcpS) localizes throughout the cytoplasm, suggesting this degradation pathway is not highly organized. In addition we find that Dcp2 is localized to P-granules, showing that Dcp2 is stored and/or active in these structures. However RNAi of these decapping enzymes has no obvious effect on embryogenesis. In contrast we find that nuclear cap binding proteins (CBP-20 and 80), eIF4G, and PAB-1 are absolutely required for development. Together our data provides further evidence that pathways of general mRNA metabolism can be remarkably organized during development, with two different decapping enzymes localized in distinct cytoplasmic domains.

Animals↗