Blastula Stage Of Development Research Articles

CFTR deficiency causes cardiac dysplasia during zebrafish embryogenesis and is associated with dilated cardiomyopathy

Mutations in the CFTR gene cause cystic fibrosis (CF) with myocardial dysfunction. However, it remains unknown whether CF-related heart disease is a secondary effect of pulmonary disease, or an intrinsic primary defect in the heart. Here, we used zebrafish, which lack lung tissue, to investigate the role of CFTR in cardiogenesis. Our findings demonstrated that the loss of CFTR impairs cardiac development from the cardiac progenitor stage, resulting in cardiac looping defects, a dilated atrium, pericardial edema, and a decrease in heart rate. Furthermore, we found that cardiac development was perturbed in wild-type embryos treated with a gating-specific CFTR channel inhibitor, CFTRinh-172, at the blastula stage of development, but not at later stages. Gene expression analysis of blastulas indicated that transcript levels, including mRNAs associated with cardiovascular diseases, were significantly altered in embryos derived from cftr mutants relative to controls. To evaluate the role of CFTR in human heart failure, we performed a genetic association study on individuals with dilated cardiomyopathy and found that the I556V mutation in CFTR, which causes a channel defect, was associated with the disease. Similar to other well-studied channel-defective CFTR mutants, CFTR I556V mRNA failed to restore cardiac dysplasia in mutant embryos. The present study revealed an important role for the CFTR ion channel in regulating cardiac development during early embryogenesis, supporting the hypothesis that CF-related heart disease results from an intrinsic primary defect in the heart.

Formation and maintenance of the organizer among the vertebrates.

The organizer is established at the blastula stage of development, under the influence of a special region of cells known as the Nieuwkoop center in amphibians, where Vg1/activin-like signals overlap with activity of the Wnt-pathway. Despite differences in their mode of early development, a similar region can be identified in other vertebrates. It has widely been assumed that once the organizer property is assigned to cells at this early stage, it is fixed so that by the gastrula stage, no new cells acquire organizer properties. However, when the organizer is ablated, it can regenerate for a limited period during gastrulation, a process regulated by both positive and negative signals emanating from various domains in the embryo. Here we compare the mechanisms that initially establish the organiser in the blastula with those that maintain it during gastrulation in different vertebrate classes, and argue that similar molecular mechanisms may be involved in the two processes. We also suggest that these mechanisms are required to ensure the appropriate location of the organizer property in the gastrula, where cells are continuously moving.

DNA replication and nuclear organization: prospects for a soluble in vitro system.

The role of nuclear structure in the replication of eukaryotic DNA has been the subject of debate for many decades. The recent demonstration that once-per-cell-cycle replication can take place in vitro without a nucleus, providing sufficiently high concentrations of replication factors are supplied, suggests that one role of the nucleus is to concentrate essential factors. This important finding has paved the way for the establishment of a purified biochemical system for replication of eukaryotic DNA. However, this soluble system, derived from Xenopus egg extracts, initiates replication within any DNA sequence and does not recapitulate the spatial and temporal regulation of DNA replication that is observed in most cells. In both Xenopus and Drosophila embryos, site-specific initiation of replication is not observed until after nuclei become transcriptionally active at the blastula stage of development. Furthermore, programmed changes in both the locations of origins and the time during S-phase at which sequences are replicated accompany key stages of metazoan development. Recent findings indicate that these changes correlate with changes in nuclear organization and that the spatial and temporal program for replication is established early in G1-phase when nuclei are structurally and functionally reorganized after mitosis.

The sea urchin egg yolk granule is a storage compartment for HCL-32, an extracellular matrix protein

We have utilized protein gel blot analysis and immunogold labelling to define the intracellular storage compartment for HCL-32, a 32-kDa protein component of the sea urchin embryonic extracellular matrices, the hyaline layer and basal lamina. Anti-HCL-32 antiserum specifically labelled yolk granules in unfertilized eggs. Cortical granules, mitochondria, sparse granules, and lipid vacuoles were not labelled. Label continued to be detected in the yolk granules through to the blastula stage of development. However, by the gastrula stage no labelling was detected in the yolk granules. In protein gel blot analysis HCL-32 was detected in yolk granules prepared from unfertilized eggs. These results clearly define the yolk granule as a storage compartment for HCL-32, an extracellular matrix protein.

Postfertilization deadenylation of mRNAs in Xenopus laevis embryos is sufficient to cause their degradation at the blastula stage.

Although the maternal Xenopus laevis Eg mRNAs are deadenylated after fertilization, they are not immediately degraded and they persist in the embryos as poly(A)- transcripts. The degradation of these RNAs is not detected until the blastula stage of development (6 to 7 h postfertilization). To understand the basis for this delay between deadenylation and degradation, it is necessary to identify the cis-acting element(s) required to trigger degradation in blastula stage embryos. To this end, several chimeric RNAs containing different portions of the 3' untranslated region of Eg2 mRNA were injected into two-cell X. laevis embryos. We observed that only the RNAs that contained the cis-acting elements that confer rapid deadenylation were subsequently degraded at the blastula stage. This suggested that deadenylation may be sufficient to trigger degradation. By injecting chimeric RNAs devoid of Eg sequence information, we further showed that only deadenylated RNAs were degraded in X. laevis embryos. Last, introduction of a functional cytoplasmic polyadenylation element into a poly(A)- RNA, thereby causing its polyadenylation after injection into embryos, protected the RNA from degradation. Hence, in X. laevis embryos, the postfertilization deadenylation of maternal Eg mRNAs is sufficient to cause the degradation of an mRNA, which, however, only becomes apparent at the blastula stage. Possible causes for this delay between deadenylation and degradation are discussed in the light of these results.

SpRunt-1, a New Member of the Runt Domain Family of Transcription Factors, Is a Positive Regulator of the Aboral Ectoderm-SpecificCyIIIAGene in Sea Urchin Embryos

In this paper we present a structural and functional characterization of a new sea urchin embryo transcription factor, SpRunt-1. This factor was isolated by means of its specific interaction with acis-regulatory target site of theCyIIIagene. Here we show that this target site, the P7I site, is required for normal embryonic activation ofCyIIIa·CATreporter gene constructs. An oligonucleotide affinity column bearing the P7I target site purifies a 21-kDa polypeptide from blastula-stage nuclear extracts, and the amino acid sequence obtained from this polypeptide was used to generate a nucleic acid probe with which the corresponding cDNA was cloned. The cDNA encodes an approximately 60-kDa protein, SpRunt-1, which includes a “runt domain” that is closely homologous to those ofDrosophilaand mammalian runt domain transcription factors. RNA and genomic blots show that SpRunt-1 is represented by a single embryonic transcript, encoded by one of possibly two runt-domain-containing genes. By RNA probe protection we found that transcripts of SpRunt-1 increase in concentration dramatically after the blastula stage of development, suggesting that the up-regulation ofCyIIIathat occurs after blastula stage is a function of zygotically transcribed SpRunt-1. These results are discussed with reference to known features of the runt domain family of transcription factors.

Purification and characterization of a 32-kDa protein that localizes to the sea urchin extraembryonic matrix, the hyaline layer.

We have purified a 32 kilodalton (kDa) protein that localized with isolated, intact hyaline layers prepared from 1-h-old embryos. The protein appeared not to bind calcium and was not quantitatively released from 1-h-old embryos in the absence of Ca2+ and Mg2+. Using polyclonal antiserum prepared against the 32-kDa protein, the antigen was detected throughout embryonic development. By the hatched blastula stage of development, the 32-kDa protein was replaced by a species of slightly smaller molecular mass. Quantitative determination indicated that the 32-kDa protein accounted for approximately 6% of the total protein present in the sea urchin egg. This result is suggestive of a structural role for the 32-kDa protein that is required throughout embryonic development, although perhaps in a modified form from the hatched blastula stage on.

Differential Compartmentalization of Plasmid DNA Microinjected into Xenopus laevis Embryos Relates to Replication Efficiency

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).

Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency.

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).

Differential replication of circular DNA molecules co-injected into early Xenopus laevis embryos.

Replication of co-injected supercoiled DNA molecules in fertilized Xenopus eggs was monitored through the blastula stage of development. The extent of replication, as measured by 32P-dTMP incorporation into form I DNA, was directly proportional to the number of molecules, rather than the size, of the plasmid injected. Although only a small fraction of molecules of either template was replicated, incorporation was predominantly into full length daughter molecules. Over at least a 20-fold concentration range of microinjected DNA, injection of equal masses of DNA resulted in greater incorporation into the smaller form I DNA present in molar excess. The extent of incorporation into supercoiled DNA for a particular plasmid was apparently independent of the concentration of a second, co-injected plasmid. The relative extents of replication of co-injected supercoiled templates could be altered simply by changing the molar ratios of the templates.

The factors that promote the development of symmetry properties in aggregates from dissociated echinoid embryos.

Embryos of Hemicentrotus pulcherrimus at the 16 cell, 400 cell or mesenchyme blastula stage of development were dissociated into single cells. The cells were reaggregated, and the development of individual aggregates was monitored. Only aggregates from 16 cell embryos developed into pluteus-like larvae with radial or bilateral symmetry. When embryos at these three developmental stages were incompletely dissociated so that there were mixtures of single cells and groups of undissociated cells, the percentage of aggregates from 16 cell embryos that developed in a pluteus-like manner was greater than in aggregates from completely dissociated 16 cell embryos. Also a small percentage of aggregates from 400 cell embryos now developed into pluteus-like larvae. In both of these experiments small aggregates tend to develop in a more normal manner than larger aggregates.In order to test the role of undissociated cells in promoting pluteus-like development in aggregates from incompletely dissociated blastula stage embryos, pieces of intact animal, lateral, or vegetal blastula wall were grafted to aggregates formed from completely dissociated embryos. While each kind of graft improved the ability of the aggregate to develop in a pluteus-like manner, grafts of vegetal blastula wall were most effective. In an aggregate, a graft differentiates according to its presumptive fate and influences the cells of the aggregate to differentiate in an appropriate manner. The ability of the graft to influence the development of the other cells in the aggregate depends on the developmental stage of the cells that make up the aggregate and the size of the aggregate.

Both basal and ontogenic promoter elements affect the timing and level of expression of a sea urchin H1 gene during early embryogenesis.

Late histone H1-beta mRNA accumulates with the correct ontogenic pattern following microinjection of the cloned gene into fertilized sea urchin eggs. Sequences upstream of the gene encoding the sea urchin H1-beta protein contain both basal and developmentally regulated elements. One late H1-specific activator sequence (USE IV) is required for the accumulation of mRNA following the blastula stage of development. All late H1 genes also contain a highly conserved GC-rich sequence resembling a low-affinity binding site for the mammalian transcription factor Sp1 that is required for basal expression of the H1-beta gene at all stages of embryogenesis. When this GC-rich sequence (GGGCTG) is converted to a perfect core Sp1 sequence (GGGCGG), the H1-beta transcripts accumulate to much greater levels and their peak accumulation is shifted to the early blastula stage rather than late blastula and gastrula stages of development. Coincidently, early H1 genes, whose peak expression is also at the early blastula stage, all contain the same core consensus sequence (GGGCGG). Thus, both gene-specific activator sequences, as well as sequences that resemble sites for general transcription factors, may play a major role in determining the temporal patterns of gene expression during early embryogenesis.

An antiserum to the sea urchin 20 S egg dynein reacts with embryonic ciliary dynein but it does not react with the mitotic apparatus

Unfertilized sea urchin eggs contain one or more dynein-like enzymes which may be able to serve as microtubule translocators during embryonic development. There are at least two interesting possibilities for the function of the egg dynein: the enzyme may be involved in cytoplasmic microtubule movement such as mitotic spindle anaphase motion; or the enzyme may be a stored precursor for the dynein that functions in embryonic cilia, which are expressed and highly motile at the blastula stage of development. In order to determine directly the distribution and possible function of one of the previously described egg dyneins, the latent-activity 20 S egg dynein ( Asai and Wilson, 1985), an antiserum was produced which was highly reactive with the important high M r polypeptides of 20 S dynein. This antiserum reacted in “Western” immunoblots and in dot-blotting experiments with egg dynein and with embryonic ciliary dynein, but it did not react with any component of sperm flagella. Indirect double immunofluorescence microscopy demonstrated that the anti-20 S antiserum could brightly stain embryonic cilia but it did not stain the sperm flagella from the same sea urchin species. Under the same conditions that the antiserum stained cilia, anti-20 S did not stain the mitotic apparatus but it did appear to stain the cortical region of the dividing egg. In a time-course experiment, the antigen reactive with the anti-20 S antiserum gradually accumulated in the developing early sea urchin embryo. The most significant increase in the apparent concentration of the 20 S dynein occurred just prior to embryonic ciliation and during a period when the mitotic activity of the embryo was in decline. These results lead to two conclusions. First, ciliary dynein and sperm flagellar dynein, although derived from very similar organelles and from the same species of sea urchin, are immunologically distinct. Second, the 20 S egg dynein may be a stored precursor of embryonic ciliary dynein and does not appear to be a component of the mitotic apparatus.

Ammonium lon as a possible regulator of the commencement of rRNA synthesis in Xenopus laevis embryogenesis

Recently, we have shown that ammonium salts and amines at an external concentration of 3 m M selectively inhibit rRNA synthesis in Xenopus disaggregated neurula cells. We studied here the change in the amount of ammonia within the embryo and its inhibitory action on the commencement of rRNA synthesis which normally occurs at the blastula stage of development. Ammonia exists at ca. 50 ng/egg (or ca. 3.0 m M at an intra-egg concentration) in the unfertilized egg. This level was maintained during cleavage and then sharply decreased during the blastula stage to the level of ca. 20 ng/embryo (or 1.2 m M) in postblastular stages. Ammonia was extracted from cleavage embryos in a form of ammonium chloride and confirmed to selectively inhibit rRNA synthesis in neurula cells. With authentic ammonium chloride, ammonia was found to be promptly incorporated into cells and to inhibit rRNA synthesis within 1 hr after treatment. In blastula cells, ammonium salts reversibly inhibited the commencement of the synthesis of rRNA, but not hnRNA, 5 S RNA and U1, U2, and U5 snRNAs. The inhibition was at the step of transcription of 40 S pre-rRNA but not the processing or degradation of the processed rRNA. Ammonium salts did not inhibit DNA synthesis, protein synthesis, cell division, and cellular reaggregation. These observations suggest that ammonium ion may be involved in the regulation of the commencement of rRNA synthesis in Xenopus embryogenesis, although it is not yet clear if the ammonium ion exerts its effect directly upon the rDNA transcription system.

Prolyl hydroxylase activity during sea urchin development

Prolyl hydroxylase activity appears at the blastula stage of development in the sea urchin Strongylocentrotus purpuratus and increases over 7-fold by the prism larva stage. The enzyme requires ascorbate, ferrous ions, and α-ketoglutarate for maximum activity and is inhibited by α,α′-dipyridyl. The significance of prolyl hydroxylase activity in embryonic collagen metabolism and morphogenesis is discussed.

THE CHROMOSOME CYCLE OF PHENACOCCUS (COCCIDÆ)

1. The diploid chromosome number of Phenacoccus acericola is twelve in both sexes. 2. The female is normal in all respects. All eggs undergo two maturation divisions with typical synapsis and reduction of the choromosomes to the haploid condition. 3. In the male, from the blastula stage of development on, one haploid set of chromosomes maintains a permanently condensed condition, forming a pycnotic knot in resting phases and separating into its component chromosomes at division. 4. Meiosis in the male is effected without synapsis. The first division is equational and diploid. Without interphase the telophase chromosomes separate into two groups of six, one condensed and closely aggregated, and one loosely arranged and more diffuse. A single half spindle forms in connection with the condensed group and reduction is effected by the consequent passage of the condensed group to the single pole of the spindle. 5. No cytoplasmic division accompanies the second division; the binucleate spermatids thus formed fuse in pairs. 6. That half of the spermatid nuclei originating from the condensed chromosome set disintegrates without trace of sperm development; the other half of the spermatid nuclei, those originating from the nonpycnotic normal chromosomes, develops into typical coccid sperm.