Biomedical subjects

E H Davidson

Publications and source records attributed to E H Davidson.

At least 109 records · Page 6Linked to original sources

PubMed1991

Differential stability of expression of similarly specified endogenous and exogenous genes in the sea urchin embryo.

The object of these experiments was to determine whether competitive titration in vivo of factors required for expression of the CyIIIa.CAT fusion gene would affect expression of the endogenous CyIIIa gene in the same embryos. Earlier work showed that expression of this fusion gene after injection into sea urchin eggs is stoichiometrically reduced when low molar excesses of DNA fragments containing only its regulatory domain are coinjected. In order to compare endogenous (i.e. CyIIIa) and exogenous (i.e. CyIIIa.CAT) expression simultaneously in embryos bearing excess competitor regulatory DNA, we developed, and here describe, a new procedure for generating transgenic sea urchin embryos in which all of the cells in many embryos, and most in others, bear the exogenous DNA. Such large reduction of mosaicism can be achieved by multiple injection of the exogenous DNA fragments into fertilized eggs. Using this method, we demonstrate that at a level of competitor DNA incorporation which reduces CyIIIa.CAT expression by 85%, endogenous CyIIIa mRNA levels are wholly unaffected. Nor is spatial expression of the endogenous CyIIIa gene disturbed. Since the CyIIIa.CAT genes are properly expressed under control of the CyIIIa regulatory sequences, they must participate in the same set of necessary DNA-protein interactions. However, we infer from the results that we report here that the regulatory complexes in the endogenous CyIIIa gene are greatly stabilized relative to those of the exogenous CyIIIa.CAT genes.

Animals↗

PubMed1991

Macromere cell fates during sea urchin development.

This paper examines the cell lineage relationships and cell fates in embryos of the sea urchin Strongylocentrotus purpuratus leading to the various cell types derived from the definitive vegetal plate territory or the veg2 tier of cells. These cell types are gut, pigment cells, basal cells and coelomic pouches. They are cell types that constitute embryonic structures through cellular migration or rearrangement unlike the relatively non-motile ectoderm cell types. For this analysis, we use previous knowledge of lineage to assign macromeres to one of four types: VOM, the oral macromere; VAM, the aboral macromere, right and left VLM, the lateral macromeres. Each of the four macromeres contributes progeny to all of the cell types that descend from the definitive vegetal plate. Thus in the gut each macromere contributes to the esophagus, stomach and intestine, and the stripe of labeled cells descendant from a macromere reflects the re-arrangement of cells that occurs during archenteron elongation. Pigment cell contributions exhibit no consistent pattern among the four macromeres, and are haphazardly distributed throughout the ectoderm. Gut and pigment cell contributions are thus radially symmetrical. In contrast, the VOM blastomere contributes to both of the coelomic pouches while the other three macromeres contribute to only one or the other pouch. The total of the macromere contribution amounts to 60% of the cells constituting the coelomic pouches.

Animals↗

PubMed1990

Sequences of the CyIIIa actin gene regulatory domain bound specifically by sea urchin embryo nuclear proteins.

Expression of the CyIIIa cytoskeletal actin gene is a marker of differential gene activation in the aboral ectoderm of the early sea urchin embryo. Gene transfer experiments have defined a 2,300 nucleotide cis-regulatory domain required for the correct spatial and temporal control of this gene. This domain includes at least 20 sites at which relatively stable DNA--protein complexes form in vitro on reaction with embryo nuclear extracts. We report the nucleotide sequence of the whole regulatory domain and map the sites at which high-specificity DNA--protein interactions occur. These were located initially by gel shift assays carried out on progressive restriction digests of given subfragments of the large regulatory domain and were located more exactly by oligonucleotide gel shift competitions. Eight of the sites of specific interaction are unique within the CyIIIa regulatory domain, and the remainder consist of five different sites that occur more than once. We observe some well known sequences also found in regulatory regions of other genes, e.g., "CCAAT" and "octamer" elements. The various sites have been classified regarding putative biological function in other work, and the present studies permit an assessment of the number and complexity of interactions constituting each functional class and of the relative locations of sites of each class.

Actins↗

PubMed1990

Rapid evolution in a fraction of the Drosophila nuclear genome.

Previous observations have indicated that Drosophila DNA contains a component that evolves so rapidly that it fails to hybridize between the DNAs of sibling species. To establish the reality of this component and study its properties, the fraction (about 20%) of Drosophila simulans (Dsim) DNA that fails to hybridize to Drosophila melanogaster (Dmel) DNA has been isolated. The majority of the hybridizable part of this isolated fraction (based on control tests on Dsim DNA) fails to hybridize with Dmel DNA under the conditions used for the initial fractionation. Clones of this fraction do hybridize with Dmel DNA at open criterion producing duplexes with greatly reduced thermal stability, indicating that the underlying process is rapid sequence divergence rather than loss of the homologous sequences by relatively large deletions. Cloned fragments from the nonhybridizing fraction from Dsim are more than 15% divergent from the Dmel homologues, whereas the fraction that does hybridize is only 3-5% divergent. In comparison, synonymous substitutions in the coding regions of five genes show a 9% average divergence between Dsim and Dmel. They appear to be intermediate in their degree of divergence between the hybridizing and nonhybridizing components.

Animals↗

PubMed1990

Segregation of oral from aboral ectoderm precursors is completed at fifth cleavage in the embryogenesis of Strongylocentrotus purpuratus.

A specific set of founder cells uniquely gives rise to the oral and aboral ectoderms in the regularly developing sea urchin Strongylocentrotus purpuratus. We showed earlier that the polar No and Na (animal oral and animal aboral) blastomeres are specified by third cleavage, while the respective oral and aboral lineage contributions of the left and right NL (animal lateral) blastomeres have not yet segregated from one another at third cleavage. Here we demonstrate by iontophoretic injection of lysyl rhodamine dextran lineage tracer that segregation of oral vs aboral cell fates in the lineages of the NL blastomeres has still not occurred by fourth cleavage, but at fifth cleavage there arise from the NL sublineages founder cells whose progeny contribute exclusively to the aboral ectoderm. The sister cells of these fifth cleavage blastomeres are founder cells that contribute exclusively to oral structures. The aboral ectoderm tracts to which NL derivatives give rise occupy lateral regions of the anterior aboral ectoderm, while the oral structures deriving from the NL blastomeres are the lateral sectors of the ciliated bands. The cells of the ciliated bands do not express aboral ectoderm markers and are considered to constitute the border of the oral region. With these new findings we complete our knowledge of the origins, identities, and fates of the 11 founder cells, the progeny of which exclusively give rise to the aboral ectoderm, and of the 5 founder cells, the progeny of which exclusively produce the oral ectoderm and its derivatives.

Animals↗

PubMed1990

Locale and level of bindin mRNA in maturing testis of the sea urchin, Strongylocentrotus purpuratus.

This first study of the onset of spermatogenesis in the sea urchin, Strongylocentrotus purpuratus, was undertaken using individuals reared in the laboratory. Spermatogenesis commences about 11-12 months after metamorphosis in these animals. Bindin message accumulates in late spermatocytes and early spermatids which lie in the luminal germinal layer. Bindin message accumulates later than does the testis-specific histone, H2b-1, suggesting that different classes of genes are sequentially activated during the differentiation of sperm. We correlate the number of bindin mRNA molecules with morphological structure and with quantitative aspects of gonad maturation including the number of nuclei and of sperm. The results suggest that the bindin mRNA concentration in total RNA from testis at different stages of maturation reflects the change in the proportion of expressing cells in the total cell population of the testis.

Animals↗

PubMed1990

Rare maternal mRNAs code for regulatory proteins that control lineage-specific gene expression in the sea urchin embryo.

The prevalence of mRNAs coding for the sea urchin embryo regulatory factors P3A1 and P3A2 was measured by single-strand probe excess solution hybridization. P3A1 and P3A2 are not homologous proteins, though they both bind specifically to a particular cis-regulatory sequence. Interaction at this target site is known to be required for lineage-specific expression of an aboral ectoderm-specific gene and probably for several other genes as well. Genome blot hybridizations show that both factors are encoded by single-copy genes. Maternal mRNAs for both factors are present at less than 10(3) molecules per egg, which places them in the rare mRNA class. During development to the mesenchyme blastula stage, the amount of P3A1 mRNA (per embryo) increases severalfold while that of P3A2 remains approximately constant. Specification of the aboral ectoderm founder cells and of their initial patterns of gene expression must occur during early to mid-cleavage stage. Therefore, the regulatory proteins needed for this process must be produced by this stage. We show that the quantities of the P3A proteins that can be synthesized from the numbers of mRNA molecules present in the large blastomeres of the early embryo are sufficient to be functional, because these proteins will be accumulated in the nuclei. Thus maternal P3A1 or P3A2 proteins asre not required, nor were these detected in earlier studies. Furthermore, differential spatial (as well as temporal) distribution of both of these newly synthesized factor species could result from the unequal cleavage pattern utilized in the sea urchin egg.

Animals↗

PubMed1990

Intersecting batteries of differentially expressed genes in the early sea urchin embryo.

We determined the distribution of cis-regulatory sites, previously identified in the control domain of the CyIIIa gene, in three other genes displaying diverse spatial patterns of expression in the sea urchin embryo. Competitive gel-shift reactions were carried out using probes from the CyIIIa gene, with competitor fragments isolated from the previously defined control domains of the other genes. CyIIIa is expressed only in aboral ectoderm lineages; the other genes studied were Spec1, also expressed in aboral ectoderm; CyI, expressed in many different cell types; and SM50, expressed only in skeletogenic mesenchyme. All four genes are activated at about the same time in late cleavage. Where competitive interactions indicated a functionally comparable binding site (in vitro), a sequence homology was sought, and in most cases could be identified. An interesting pattern of putative regulatory site usage emerges: Of 10 CyIIIa interactions tested, three only were unique to the CyIIIa gene with respect to the set of four genes tested; one believed on previous evidence to be a temporal regulator was shared by all four genes, and the remainder were shared in various subsets of the four genes.

Animals↗

PubMed1990

How embryos work: a comparative view of diverse modes of cell fate specification.

Embryonic processes in the nematode C. elegans, the gastropod mollusc Ilyanassa, the dipteran Drosophila, the echinoid Strongylocentrotus purpuratus, the ascidian Ciona, the anuran Xenopus, the teleost Brachydanio and mouse are compared with respect to a series of parameters such as invariant or variable cleavage, the means by which the embryonic axes are set up, egg anisotropies and reliance on conditional or on autonomous specification processes. A molecular interpretation of these modes of specification of cell fate in the embryo is proposed, in terms of spatial modifications of gene regulatory factors. On this basis, classically defined phenomena such as regulative development and cytoplasmic localization can be interpreted at a mechanistic level, and the enormous differences between different forms of embryogenesis in the Animal Kingdom can be considered within a common mechanistic framework. Differential spatial expression of histospecific genes is considered in terms of the structure of the gene regulatory network that will be required in embryos that utilize cell-cell interaction, autonomous vs conditional specification and maternal spatial information to differing extents. It is concluded that the regulatory architectures according to which the programs of gene expression are organized are special to each form of development, and that common regulatory principles are to be found only at lower levels, such as those at which the control regions of histospecific structural genes operate.

Animals↗

PubMed1990

Competitive titration in living sea urchin embryos of regulatory factors required for expression of the CyIIIa actin gene.

Previous studies have located some twenty distinct sites within the 2.3 kb 5' regulatory domain of the sea urchin CyIIIa cytoskeletal actin gene, where there occur in vitro high-specificity interactions with nuclear DNA-binding proteins of the embryo. This gene is activated in late cleavage, exclusively in cells of the aboral ectoderm cell lineages. In this study, we investigate the functional importance in vivo of these sites of DNA-protein interaction. Sea urchin eggs were coinjected with a fusion gene construct in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene is under the control of the entire CyIIIa regulatory domain, together with molar excesses of one of ten nonoverlapping competitor subfragments of this domain, each of which contains one or a few specific site(s) of interaction. The exogenous excess binding sites competitively titrate the available regulatory factors away from the respective sites associated with the CyIIIa.CAT reporter gene. This provides a method for detecting in vivo sites within the regulatory domain that are required for normal levels of expression, without disturbing the structure of the regulatory domain. We thus identify five nonoverlapping regions of the regulatory DNA that apparently function as binding sites for positively acting transcriptional regulatory factors. Competition with a subfragment bearing an octamer site results in embryonic lethality. We find that three other sites display no quantitative competitive interference with CyIIIa.CAT expression, though as shown in the accompanying paper, two of these sites are required for control of spatial expression. We conclude that the complex CyIIIa regulatory domain must assess the state of many distinct and individually necessary interactions in order to properly regulate CyIIIa transcriptional activity in development.

Actins↗

PubMed1990

Negative spatial regulation of the lineage specific CyIIIa actin gene in the sea urchin embryo.

The CyIIIa.CAT fusion gene was injected into Strongylocentrotus purpuratus eggs, together with excess ligated competitor sequences representing subregions of the CyIIIa regulatory domain. In this construct, the chloramphenicol acetyltransferase (CAT) reporter gene is placed under the control of the 2300 nucleotide upstream regulatory domain of the lineage-specific CyIIIa cytoskeletal actin gene. CAT mRNA was detected by in situ hybridization in serial sections of pluteus stage embryos derived from the injected eggs. When carrier DNA lacking competitor CyIIIa fragments was coinjected with CyIIIa.CAT, CAT mRNA was observed exclusively in aboral ectoderm cells, i.e. the territory in which the CyIIIa gene itself is normally expressed (as also reported by us previously). The same result was obtained when five of seven different competitor subfragments bearing sites of DNA-protein interaction were coinjected. However, coinjection of excess quantities of either of two widely separated, nonhomologous fragments of the CyIIIa regulatory domain produced a dramatic ectopic expression of CAT mRNA in the recipient embryos. CAT mRNA was observed in gut, mesenchyme cells and oral ectoderm in these embryos. We conclude that these fragments contain regulatory sites that negatively control spatial expression of the CyIIIa gene.

Actins↗

PubMed1989

In vivo competition identifies positive cis-regulatory elements required for lineage-specific gene expression in the sea urchin embryo.

Several cis-regulatory elements within the 5' regulatory region of the lineage-specific CyIIIa actin gene have been identified by in vivo competition. Sea urchin eggs were coinjected with a fusion construct in which the bacterial chloramphenicol acetyltransferase (CAT) gene is controlled by the CyIIIa regulatory domain, together with molar excesses of various DNA subfragments that are derived from this region. Each subfragment studied includes one or several known sites where highly specific interactions occur in vitro with nuclear DNA-binding proteins. Coinjection of excess molecules of some of these subregions results in a decrease in the activity of the CyIIIa-CAT fusion gene, as a function of the molar subfragment: CyIIIa-CAT ratio. This result implies that these sites complete with cis sequences linked to the CAT reporter gene for limited factors that positively regulate CyIIIa transcription in the embryo, and demonstrates the functional importance of a number of the DNA-protein interactions that have been observed in vitro.

Actins↗

PubMed1989

Expression of two actin genes during larval development in the sea urchin Strongylocentrotus purpuratus.

We report the first measurements of cell number, total RNA, and transcript accumulations for two actin genes during larval development of the sea urchin Strongylocentrotus purpuratus. At 5 weeks of feeding, when development of laboratory-raised larvae is completed, the cell number has increased about 100-fold with respect to the pluteus-stage embryo to about 150,000 +/- 50,000, and the total RNA has increased 46-fold to about 130 ng per larva. The transcripts of the Cylla cytoskeletal actin gene, which is expressed in adult tissues, continue to accumulate throughout larval development. A contrasting pattern of transcript accumulation is observed for Cyllla, a different cytoskeletal actin gene that in the embryo is expressed only in aboral ectoderm. These transcripts increase in number early in larval development, when the larval epidermis is differentiating, and then decline in quantity. It is known that at metamorphosis the larval epidermis is largely histolyzed and that the Cyllla gene is not expressed in the juvenile or adult.

Actins↗

PubMed1989

The current source of human Alu retroposons is a conserved gene shared with Old World monkey.

A significant fraction of human Alu repeated sequences are members of the precise, recently inserted class. A cloned member of this class has been used as a probe for interspecies hybridization and thermal stability determination. The probe was reassociated with human, mandrill, and spider monkey DNA under conditions such that only almost perfectly matching duplexes could form. Equally precise hybrids were formed with human and mandrill DNA (Old World monkey) but not with spider monkey DNA (New World). These measurements as well as reassociation kinetics show the presence in mandrill DNA of many precise class Alu sequences that are very similar or identical in quantity and sequence to those in human DNA. Human and mandrill are moderately distant species with a single-copy DNA divergence of about 6%. Nevertheless, their recently inserted Alu sequences arise by retroposition of transcripts of source genes with nearly identical sequences. Apparently a gene present in our common ancestor at the time of branching was inherited and highly conserved in sequence in both the lineage of Old World monkeys and the lineage of apes and man.

Animals↗

PubMed1989

Lineage-specific gene expression and the regulative capacities of the sea urchin embryo: a proposed mechanism.

Three aspects of early sea urchin development are reviewed, and conclusions derived that lead to a unified concept of how the initial specifications of differential gene activity may occur in this embryo. i. The embryo has an invariant cell lineage, and the lineage founder cells can be considered as regulatory spatial domains. That is, from each of these cells descend clones of progeny the members of which express the same set of lineage-specific genes. ii. From the extensive classical literature on blastomere plasticity, and some key modern experiments, are derived a system of inductive blastomere interactions, which accounts for the conditionality of lineage founder cell specification. That is, the fates of many of the lineage founder cells can apparently be altered if the normal spatial interrelationships within the embryo are perturbed. iii. Recent studies have been carried out by gene transfer, and are supported by in vitro analyses of DNA-protein interactions in the regulatory regions of two genes that are expressed in a lineage- specific manner. Expression of both of these markers of cell fate specification is controlled by diffusible DNA-binding factors (i.e. within each nucleus). A molecular mechanism is proposed, based on inductive effects on gene regulatory factors, which in principle provides a specific explanation of the regulative capacities for which this embryo is famous.

Animals↗

PubMed1989

The oral-aboral axis of a sea urchin embryo is specified by first cleavage.

Several lines of evidence suggest that the oral-aboral axis in Strongylocentrotus purpuratus embryos is specified at or before the 8-cell stage. Were the oral-aboral axis specified independently of the first cleavage plane, then a random association of this plane with the blastomeres of the four embryo quadrants in the oral-aboral plane (viz. oral, aboral, right and left) would be expected. Lineage tracer dye injection into one blastomere at the 2-cell stage and observation of the resultant labeling patterns demonstrates instead a strongly nonrandom association. In at least ninety percent of cases, the progeny of the aboral blastomeres are associated with those of the left lateral blastomeres and the progeny of the oral blastomeres with the right lateral ones, respectively. Thus, ninety percent of the time the oral pole of the future oral-aboral axis lies 45 degrees clockwise from the first cleavage plane as viewed from the animal pole. The nonrandom association of blastomeres after labeling of the 2-cell stage implies that there is a mechanistic relation between axis specification and the positioning of the first cleavage plane.

Animals↗

PubMed1988

Mosaic incorporation and regulated expression of an exogenous gene in the sea urchin embryo.

A fusion gene construct in which the bacterial chloramphenicol acetyltransferase (CAT) gene is controlled by CyIIIa actin gene cis-regulatory sequences was injected into unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. The distribution of CAT DNA sequences was measured directly by in situ hybridization in squashed 24-hr blastula preparations derived from these eggs. Earlier studies had shown that stable mosaic incorporation of the exogenous DNA occurs during cleavage, after which the exogenous sequences replicate at approximately the pace of the host cell genomes. The fractions of embryonic cells observed in this study to include CAT DNA sequences imply that their stable incorporation into a replicating nuclear form occurs most often in a single cell at the 3rd or 4th cleavage stages, though it may occur as early as 2nd cleavage, or as late as 7th cleavage. Corroborative measurements were carried out by the same method on squashed preparations of embryos at earlier stages, and by in situ hybridizations of CAT mRNA, both in dissociated embryos and in cytological sections of 72-hr pluteus-stage embryos. Hybridizations to CAT mRNA and to CAT DNA were carried out on alternate sections of several embryos. The results confirm unequivocally that although CAT mRNA appears only in the aboral ectoderm in embryos derived from eggs injected with the CyIIIa.CAT fusion gene, the exogenous sequences are indeed present, though silent, in the various other cell types of the late embryo.

Acetyltransferases↗

PubMed1988

Sources and evolution of human Alu repeated sequences.

Alu repeated sequences arising in DNA of the human lineage during about the last 30 million years are closely similar to a modern consensus. Alu repeats arising at earlier times share correlated blocks of differences from the current consensus at diagnostic positions in the sequence. Using these 26 positions, we can recognize four subfamilies and the older ones are each successively closer to the 7SL sequence. It appears that there has existed a series of conserved genes that are the primary sources of the Alu repeat family, presumably through retroposition. These genes have probably replaced each other in overlapping relays during the evolution of primates.

Animals↗