Two distinct yolk lipoprotein complexes from Caenorhabditis elegans.

The four yolk polypeptides of the nematode Caenorhabditis elegans are found in two types of lipoprotein particle: 12 S particles with Mr estimated at 450,000 and 8 S particles with Mr estimated at 250,000. Both types of particle contain approximately 8% phospholipids, 3% triglycerides, and 3% other lipids by mass. All four C. elegans yolk polypeptides can be found in either 12 or 8 S particles, depending upon the conditions of isolation. While the properties of the 12 and 8 S lipoprotein particles are consistent with a dimermonomer relationship, the asymmetric distribution of the yolk polypeptides between 12 and 8 S fractions suggests that at least two different oligomeric lipoprotein complexes are present in C. elegans embryos. In order to clarify the subunit composition of the C. elegans yolk lipoproteins, the patterns of polypeptides retained in immunoaffinity binding procedures by immunoglobulins of different antigenic specificities have been compared. When immunoaffinity binding is performed in the absence of sodium dodecyl sulfate, three C. elegans yolk proteins (yp170A, yp115, and yp88) are retained together by polyclonal immunoglobulins directed against either yp115 or yp88. A monoclonal immunoglobulin also retains these three proteins together. In contrast, a second monoclonal immunoglobulin retains only the fourth yolk protein (yp170B). Aggregate species, evidently reflecting the spontaneous formation of interchain disulfide bonds, indicate that yp170A and yp88 are physically associated, whereas yp170B self-associates in dimers. It is concluded that there are two distinct lipoprotein complexes in C. elegans: the A complex, which consists of yp170A, yp115, and yp88 and is essentially heterodimeric and the B dimer, a simple dimer of yp170B.

Posterior localization of vasa protein correlates with, but is not sufficient for, pole cell development.

The protein product of the Drosophila maternal-effect posterior group gene vasa is localized to the posterior pole of the oocyte and is sequestered by the pole cells as they form. It is, however, present at easily detectable levels throughout the oocyte and pre-blastoderm embryo. The protein is present in the pole cells and their germ line derivatives throughout all stages of development. An antiserum against this protein recognizes a pole-cell-specific antigen in seven other Drosophila species. Of six other maternal-effect loci essential for embryonic pole cell development, none affects expression of vasa, mutations in four abolish vasa protein localization, and mutations in two, tudor and valois, have little, if any, effect on vasa expression or localization. This indicates that vasa protein, when properly localized, is not sufficient for induction of pole cell development, and that at least the tudor and valois wild-type functions are also required specifically for this process. These results are discussed with respect to the multiple functions of the vasa gene.

P-element-mediated enhancer detection allows rapid identification of developmentally regulated genes and cell specific markers in Drosophila.

We have employed a new technique in Drosophila that allows in vivo detection of genomic regulatory elements using a beta-galactosidase reporter gene. A translational fusion of the reporter gene to the P-transposase gene, which is encoded by the P-transposon of Drosophila, places the expression of beta-galactosidase under the control of the weak P-transposase promoter. Flies carrying single insertions of this P-element construct at different locations in the Drosophila genome frequently stain for beta-galactosidase activity in a temporally and spatially restricted fashion in embryos, larvae and adult ovaries, reflecting the influence of nearby genomic regulatory elements on the P-transposase promoter. This technique is a powerful tool as it can be used to produce very many different cell markers and to isolate developmentally regulated genes in Drosophila. We discuss the implications of our results and the applications of the technique to further the study of Drosophila development.

Gangliosides of sea urchin embryos. Their localization and participation in early development.

The influence of antibodies to gangliosides of sea urchin Strongylocentrotus intermedius eggs on early embryos of this species was studied. gamma-Globulins were isolated from rabbit anti-ganglioside serum by micropreparative electrophoresis. These gamma-globulins produced anomalies in the development of embryos permeabilized in Triton X-100. The anomalies were not observed when anti-ganglioside gamma-globulins were added to the incubation medium together with gangliosides or when the permeabilized embryos were incubated with gamma-globulins of normal rabbit serum. Pretreatment of S. intermedius embryos with serotonin, tryptamine or some other indole derivatives led to the disappearance of ganglioside determinants from the cell surface and sharply increased immunofluorescence within the cell. Such pretreatment of embryos increased the amount of cell-associated gangliosides more than threefold as compared to untreated embryos. Serotonin was shown to bind specifically to sea urchin gangliosides immobilized on octyl-Sepharose. These observations suggest that cell-surface gangliosides, after binding drugs, are internalized and that serotonin and its antagonists inhibit the transport of newly synthesized gangliosides to the cell-surface membrane.

Interference with function of a homeobox gene in Xenopus embryos produces malformations of the anterior spinal cord.

XIHbox 1 is expressed in a narrow band across the cervical region of Xenopus embryos. The gene produces two related proteins: "long" and "short" XIHbox 1 homeodomain proteins. Injection of antibodies to the long XIHbox 1 protein into 1-cell embryos caused a phenotype in which the anterior spinal cord was morphologically transformed into a hindbrain-like structure. This alteration was restricted to the region normally expressing long XIHbox 1 protein. Injection of long protein mRNA disrupted segmentation and tissue organization without inhibiting cell proliferation. Injection of short protein mRNA into 1-cell embryos produced spinal cord malformations similar, but not identical, to those caused by the antibodies, suggesting antagonistic roles for long and short XIHbox 1 proteins. We immunostained tadpoles carrying extended hindbrains for N-CAM and consistently found defective organization of spinal nerves over the affected region.

The molecular genetics of the bithorax complex of Drosophila: characterization of the products of the Abdominal-B domain.

In Drosophila the Abdominal-B (Abd-B) domain of the bithorax complex specifies the identities of several posterior abdominal segments, comprises homeo-protein-coding regions and cis-regulatory regions, and extends from infra-abdominal-5 (iab-5) to iab-8, inclusive. Mutations that eliminate the Abd-B domain act as late embryonic lethals and result in transformations of posterior abdominal segments toward more anterior ones. The Abd-B domain gives rise to a minimum of five homeo-box-containing transcripts, 7.8, 4.7, 4.3, 3.7, and 3.3 kb in length. We examined the structure of the Abd-B domain by sequencing two Abd-B cDNA clones derived from the 4.3- and the 4.7-kb transcripts and the corresponding genomic DNA. The domain spans approximately 100 kb and contains at least eight exons. The 4.7- and 4.3-kb transcripts contain an open reading frame capable of encoding a 54-kD protein. A portion of the deduced protein-coding sequence common to all of the Abd-B transcripts was cloned into an expression vector. The resultant fusion protein then was used to derive a monoclonal antibody specific to Abd-B. By use of that antibody, we identified two embryonic Abd-B proteins, 54 and 36 kD and determined the sum of their segmental distribution by immunohistochemical analysis of whole-mounted embryos and immunofluorescent analysis of dissected embryonic nervous systems. The proteins are distributed in the fourth to the ninth abdominal segments [parasegments (PS) 10-15] inclusive. Embryos homozygous for Polycomb (Pc) show labeling over almost the entire embryo, whereas embryos deficient for the Abd-B domain show no detectable labeling.

Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector.

A P-element vector has been constructed and used to generate lines of flies with single autosomal P-element insertions. The lines were analyzed in two ways: (1) the identification of cis-acting patterning information within the Drosophila genome, as revealed by a lacZ reporter gene within the P element, and (2) the isolation of lethal mutations. We examined 3768 independent lines for the expression of lacZ in embryos and looked among these lines for lethal mutations affecting embryonic neurogenesis. This type of screen appears to be an effective way to find new loci that may play a role in the development of the Drosophila nervous system.

P-element-mediated enhancer detection: a versatile method to study development in Drosophila.

We generated and characterized greater than 500 Drosophila strains that carry single copies of a novel P-element enhancer detector. In the majority of the strains, the beta-galactosidase reporter gene in the P-transposon responds to nearby transcriptional regulatory sequences in the genome. A remarkable diversity of spatially and temporally regulated staining patterns is observed in embryos carrying different insertions. We selected numerous strains as markers for different embryonic organs, tissues, and cells. Many of these strains should allow the study of complex developmental processes, such as nervous system development, which have not been convenient to analyze previously. Also, we present genetic evidence that some of the detected regulatory elements control nearby Drosophila genes. In light of our results, we discuss the diversity and complexity of cis-acting regulatory elements in the genome and the general applications of the enhancer detector method for the study of Drosophila development.

P-element-mediated enhancer detection: an efficient method for isolating and characterizing developmentally regulated genes in Drosophila.

We describe a new approach for identifying and studying genes involved in Drosophila development. Single copies of an enhancer detector transposon, P[1ArB], have been introduced into flies at many different genomic locations. The beta-galactosidase reporter gene in this construct is influenced by a wide range of genomic transcriptional regulatory elements in its vicinity. Our results suggest that a significant proportion of these regulatory sequences are control elements of nearby Drosophila genes. These genes need not be disrupted for their regulatory elements to be identified by P[1ArB]. The P[1ArB] transposon has been designed to facilitate both rapid cloning and deletion analysis of genomic sequences into which it inserts. Therefore, the enhancer detection system is an efficient method of screening for genes primarily on the basis of their expression pattern and then rapidly analyzing those of particular interest at the molecular and genetic levels.

Immunological studies on gamma crystallins from Xenopus: localization, tissue specificity and developmental expression of proteins.

In an effort to understand the spatio-temporal regulation of crystallins and their genes during lens development, the gamma crystallins from the frog lens have been isolated, purified and characterized. Using an immunological approach, they were found to be localized exclusively in the lens fiber cells and were not detected in any other lens cells or non-lens tissues including mature oocytes. During embryogenesis, the antigens were first detected in stage 25 embryos (but not in stage 20 embryos). Their level first decreased and then increased during subsequent stages of development. A different member of the family was also found to be expressed during later stages of embryogenesis.

Effects of 2-methoxyethanol on fetal development, postnatal behavior, and embryonic intracellular pH of rats.

The industrial solvent 2-methoxyethanol (2ME) is a reproductive and developmental toxicant when administered by inhalation, gavage, and IP injection. The present research established that this solvent can produce teratogenicity in rats when administered in liquid diet. Groups of 10 Sprague-Dawley rats were given various percentages of 2ME in liquid diet on gestation days 7-18. Day 20 fetuses were examined for visceral or skeletal malformations. Concentrations above 0.025% 2ME (approximately 73 mg/kg/day) produced total embryo-mortality. Cardiovascular malformations were produced at lower levels. The teratogenic no-effect level was 0.006% 2ME (16 mg/kg). In a second experiment, groups of 12 Sprague-Dawley rats were given 0, 0.006 and 0.012% of 2ME as above. Litters were culled to 8 pups, and tested for auditory and tactile startle and conditioned lick suppression, and for performance in figure-8 activity and the Cincinnati water maze on postnatal days 48-65. The high dose of 2ME produced approximately 50% mortality in the offspring and increased the number of errors in the Cincinnati maze. No other behavioral effects were observed at either dose. An interaction study was conducted to determine if simultaneous exposure to 2ME and ethanol would reduce the teratogenicity of 2ME, but no reduction was observed. The hypothesis that 2ME acts by altering embryonic intracellular pH was tested by injecting 0.33 ml/kg of 2ME into rats on gestation day 13, and determining embryonic intracellular pH at 2, 4, 8, and 24 hours thereafter. There was an increase in pH at 4 hours, but not at later time points. Another group of rats was given 2ME along with amiloride, which blocks the sodium/hydrogen antiporter. The combined 2ME-amiloride exposure produced an incidence of cardiovascular malformations in fetuses twice that of 2ME alone. These studies confirmed the structural teratogenicity of 2ME even when given in liquid diet, as it was given for the first time in the present study. At nonteratogenic doses, developmental toxicity (e.g., postnatal deaths) persisted, but only limited evidence of behavioral teratogenicity was observed. The pH data are consistent with the concept that 2ME may alter embryonic intracellular pH at critical stages of organogenesis.

Effect of reduced protein synthesis on the cell cycle in sea urchin embryos.

We have reinvestigated the existence of cyclical fluctuations of protein synthesis and have examined the effects of reducing it in early embryos of the purple sea urchin, Strongylocentrotus purpuratus. The results show that protein synthesis increases linearly during the first 45-60 minutes after fertilization, then transiently decreases during mitosis, and rises again at first cleavage. Reducing protein synthesis of embryos to 35% its normal value only slightly affects the rate of progression through the cell cycle. It is also shown that the observed retardations of the cell cycle, under depressed protein synthesis, are attributable (by 80%) to a lengthening of the premitotic phase but also, to a lesser extent (20%), to a lengthening of the mitotic phase itself. These results suggest that mitotic proteins, in sea urchin embryos, are stable and little affected by an imposed decrease of protein synthesis during their accumulation phase. This analysis supports the view that specific mechanisms, other than decreased protein synthesis, need be turned on only at appropriate times during the cell cycle in order to explain the destruction or deactivation of mitotic proteins. Finally, a one-dimensional SDS-PAGE analysis of synthesized proteins, labeled with 35S-methionine, reveals the presence of a 50-kDa cyclin showing the expected characteristics of mitotic proteins deduced from our results.

Stage- and tissue-specific expression of two homeo box genes in sea urchin embryos and adults.

We report the isolation of two different homeo box genes, HB3 and HB4, from the Hawaiian sea urchin Tripneustes gratilla. DNA sequencing revealed a definitive Antennapedia (Antp) class homeo box in each gene. Southern transfer hybridizations showed the genes to be single-copy. A 5.7-kb transcript of the HB3 gene was found in ovary, testis, small intestine and gastrula poly(A)+ RNA. The HB4 gene produces three transcripts. A 3.7-kb and a 4.4-kb transcript are expressed during embryogenesis. A 3.5-kb transcript appears in each of the adult tissues studied. The HB4 gene appears to be the sea urchin cognate of the Drosophila infrabdominal-7 (iab-7) gene, the mouse Hox 1.7 and Hox 3.2 genes and the Xenopus X1Hbox 6 gene. An examination of Antp class homeo box genes in deuterostomes indicates that a chromosomal duplication has taken place in the evolutionary line leading to the vertebrates after the divergence of the echinoderms. Thus, the sea urchin represents the primitive condition.

Drosophila basement membrane procollagen IV. I. Protein characterization and distribution.

A collagen was isolated from Drosophila E85, Schneider line 2L and Kc cell cultures. The purified protein was characterized and antibodies were raised against it. Immunofluorescence microscopy locates this material to the regions of basement membranes of Drosophila embryos, larvae, and adults. The molecules are mostly, or entirely, homotrimers of one polypeptide chain linked by interchain disulfide bonds. The partial amino acid sequences of a cyanogen bromide cleavage product of this chain are identical with a part of the virtual translation product of the Drosophila pro alpha 1(IV) nucleotide sequence that is reported in the accompanying paper. This gene is at Drosophila chromosome location 25C and was identified by the high homology of one part of it with the noncollagenous carboxyl terminus (NC1) of vertebrate type IV basement membrane collagens (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). In the electron microscope each molecule appears as a thread with a knob at one end, which contains the carboxyl peptide domains. The variation of flexibility of the thread was mapped along its length. Pulse-chase labeling of cell cultures showed that these molecules associate into disulfide-linked dimers and higher oligomers that can be partly separated by velocity sedimentation and are resolved by sodium dodecyl sulfate-agarose gel electrophoresis. Dimers and higher oligomers formed by overlap of the amino ends of molecules were found. Mild pepsin digestion of Drosophila embryos and larvae solubilized the corresponding disulfide-linked collagen molecules, and Staphylococcus aureus V8 protease peptide maps showed the identity of the collagen derived from animals and from cell cultures. Individual, native molecules have a sedimentation coefficient s20,w = 4.1 S, the dichroic spectrum and amino acid composition of a collagen, and a Tm = 31 degrees C. Positive in situ hybridization with a specific probe for this collagen began 6-8 h after egg laying and showed message in the locations of embryos and larvae which reacted with the antibodies. This included some prominent individual cells in the hemolymph.

The distribution of nucleoplasmin in early development and organogenesis of Xenopus laevis.

The fate of the germinal vesicle-derived protein, nucleoplasmin, was followed in embryos and tadpoles of Xenopus using monoclonal antibodies and indirect immunofluorescent staining. Nucleoplasmin was found in all nuclei up to feeding tadpole stages. Thereafter its level decreased in all nuclei. It was not detected in nuclei of advanced tadpoles or of adults. Contrasting with another protein, N1, that was previously monitored in the nuclei of dividing gonia of both sexes, nucleoplasmin was only detected in the nuclei of ovarian oocytes starting at diplotene. Traces of nucleoplasmin have also been found in a rapidly-dividing fibroblastic cell-line by immunohistology and protein blotting.

The dorsal protein is distributed in a gradient in early Drosophila embryos.

dorsal is one of the maternally active dorsal-ventral polarity genes of Drosophila and is closely related to the vertebrate proto-oncogene c-rel. Genetic experiments suggest that dorsal represents one of the last (if not the last) steps in the maternal pathway involved in establishing dorsal-ventral polarity in the early embryo. Even though the dorsal RNA is uniformly distributed in the embryo, we have found that the dorsal protein is specifically localized in peripheral nuclei of syncytial and cellular blastoderm stage embryos, and it is distributed in a ventral-to-dorsal gradient. These findings suggest possible mechanisms for how the dorsal protein may communicate maternal positional information to the zygotic genome.

Vimentin expression in oocytes, eggs and early embryos of Xenopus laevis.

Immunocytochemical studies using a monoclonal anti-porcine vimentin antibody reveal a well-organized pattern of staining in Xenopus laevis oocytes, eggs and early embryos. The positions of Xenopus vimentin and desmin in two-dimensional (2D) polyacrylamide gels were first established by immunoblotting of muscle Triton extracts with anti-intermediate filament antibodies (anti-IFA), which cross-react with all intermediate filament proteins (IFPs). The anti-porcine vimentin reacts with vimentin and desmin in muscle 2D immunoblots, but only reacts with one polypeptide in oocyte blots in the position predicted for vimentin (Mr 55 x 10(3), pI 5.6). Using an anti-sense probe derived from a Xenopus vimentin genomic clone in RNase protection assays, we show that expression of vimentin begins in previtellogenic oocytes. The level of expression remains constant throughout oogenesis and in unfertilized eggs. These data suggest that vimentin is expressed in oocytes and eggs. Most interestingly, the immunocytochemical results also show that vimentin is present in the germ plasma of oocytes, eggs and early embryos. It is therefore possible that vimentin has an important role in the formation or behaviour of early germ line cells.

Formation of visual pigment chromophores during the development of Xenopus laevis.

Retinoids in the eyes of Xenopus laevis at several developmental stages, were analyzed by high-performance liquid chromatography (HPLC). At stage 37/38, larval eyes contained mainly all-trans isomers of retinal, 3-dehydroretinal, retinyl ester and 3-dehydroretinyl ester. Ratios of all-trans 3-dehydroretinal to retinal and of all-trans 3-dehydroretinyl ester to retinyl ester were almost 1 at the stage. With the advance of development, the amounts of all-trans retinal and 3-dehydroretinal decreased; however, those of all-trans retinyl ester and 3-dehydroretinyl ester increased. The chromophores of visual pigments, 11-cis retinal and 3-dehydroretinal, were detected at stage 40 (total; 0.2 pmol/eye) and their amounts increased after that stage. The ratio of 11-cis 3-dehydroretinal to retinal was almost 1 at stages 40-42. The ratio became larger after stage 43 and was almost 19 at stage 46. The ratio of all-trans 3-dehydroretinyl ester to retinyl ester, also, increased after stage 42 and reached 11 at stage 46. The mechanism of 11-cis formation during development is discussed in relation to retinoid conversions in the eyes.

A zebrafish homeobox-containing gene with embryonic transcription.

A genomic library of zebrafish (Brachydanio rerio) was constructed and screened with homeobox-containing probes. One of the strongly cross-hybridizing clones was characterized by DNA sequencing. The deduced amino acid sequence exhibits extensive homology (greater than 80%) relative to the Antennapedia-class of Drosophila homeobox sequences. Characterization of the gene with respect to expression demonstrated that two transcripts of 2.1 and 1.4 kb, respectively, are present in embryonic poly (A+) RNA. The highest concentration of the two RNA species was observed in embryos which have terminated the process of somite formation.