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

Development ◽  
1990 ◽  
Vol 110 (1) ◽  
pp. 41-50 ◽  
Author(s):  
B.R. Hough-Evans ◽  
R.R. Franks ◽  
R.W. Zeller ◽  
R.J. Britten ◽  
E.H. Davidson
Keyword(s):  
Fusion Gene ◽  
Ectopic Expression ◽  
Actin Gene ◽  
Regulatory Domain ◽  
Spatial Regulation ◽  
Sea Urchin Embryo ◽  
Oral Ectoderm ◽  
Dna Protein Interaction ◽  
Mesenchyme Cells

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.

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

Development ◽  
1990 ◽  
Vol 110 (1) ◽  
pp. 31-40 ◽  
Author(s):  
R.R. Franks ◽  
R. Anderson ◽  
J.G. Moore ◽  
B.R. Hough-Evans ◽  
R.J. Britten ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Sea Urchin ◽  
Binding Sites ◽  
Nuclear Dna ◽  
Fusion Gene ◽  
Actin Gene ◽  
Regulatory Domain ◽  
Regulatory Factors

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.

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

1990 ◽  
Vol 25 (2) ◽  
pp. 110-122 ◽  
Author(s):  
Nadine Thézé ◽  
Frank J. Calzone ◽  
Pierre Thiebaud ◽  
Ronald L. Hill ◽  
Roy J. Britten ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Nuclear Proteins ◽  
Actin Gene ◽  
Regulatory Domain ◽  
Sea Urchin Embryo ◽  
Gene Regulatory

Three Strongylocentrotus purpuratus actin genes show correct cell-specific expression in hybrid embryos of S. purpuratus and Lytechinus pictus

Development ◽  
1989 ◽  
Vol 105 (2) ◽  
pp. 407-413 ◽  
Author(s):  
P.E. Nisson ◽  
L.E. Dike ◽  
W.R. Crain
Keyword(s):  
Strongylocentrotus Purpuratus ◽  
Cell Lineage ◽  
Specific Expression ◽  
Spatial Expression ◽  
Lytechinus Pictus ◽  
Actin Genes ◽  
Oral Ectoderm ◽  
Mesenchyme Cells ◽  
Small Clusters

The cell-specific expression of three actin genes from the sea urchin species Strongylocentrotus purpuratus was examined in hybrid embryos of S. purpuratus and another species, Lytechinus pictus, by in situ hybridization. The mRNAs from each of these genes displayed distinct spatial patterns of expression in late-stage hybrid embryos (constructed in either direction), being detected only in the cell lineages where they are normally found in S. purpuratus embryos (i.e. CyIIIa, only in the aboral ectoderm lineage; CyI, in the gut, oral ectoderm and some mesenchyme cells of plutei, and preferentially in the archenteron of gastrulae; M, only in two small clusters of cells near the esophagus in plutei). These results, together with our previous observation that expression of each of these genes is activated at the same stage in these hybrid embryos as in normal S. purpuratus embryos, demonstrate that the trans-acting factors which are necessary to regulate both the temporal and spatial expression of these genes are present in the hybrid embryos. Previous experiments have shown that the expression of a chimeric gene containing the CyIIIa promoter fused to a bacterial chloramphenicol actetyltransferase (CAT) gene is not confined to the correct cell lineage (aboral ectoderm) when injected into Lytechinus embryos. The conclusion from these sets of data is that the factor(s) that regulate the spatial expression of at least one of the actin genes must derive from transcription of the zygotic genome.

scholarly journals Complexity of Developmental Control: Analysis of Embryonic Cell Lineage Specification in Caenorhabditis elegans Using pes-1 as an Early Marker

Genetics ◽  
1999 ◽  
Vol 151 (1) ◽  
pp. 131-141
Author(s):  
Laurent Molin ◽  
Heinke Schnabel ◽  
Titus Kaletta ◽  
Richard Feichtinger ◽  
Ian A Hope ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Expression Pattern ◽  
Fusion Gene ◽  
Ectopic Expression ◽  
Gene Expression Pattern ◽  
Embryonic Cell ◽  
Control Analysis ◽  
Normal Pattern ◽  
Variable Expression ◽  
Founder Cells

Abstract In the early Caenorhabditis elegans embryo five somatic founder cells are born during the first cleavages. The first of these founder cells, named AB, gives rise to 389 of the 558 nuclei present in the hatching larva. Very few genes directly involved in the specification of the AB lineage have been identified so far. Here we describe a screen of a large collection of maternal-effect embryonic lethal mutations for their effect on the early expression of a pes-1::lacZ fusion gene. This fusion gene is expressed in a characteristic pattern in 14 of the 32 AB descendants present shortly after the initiation of gastrulation. Of the 37 mutations in 36 genes suspected to be required specifically during development, 12 alter the expression of the pes-1::lacZ marker construct. The gene expression pattern alterations are of four types: reduction of expression, variable expression, ectopic expression in addition to the normal pattern, and reduction of the normal pattern together with ectopic expression. We estimate that ∼100 maternal functions are required to establish the pes-1 expression pattern in the early embryo.

Localization and expression of msp130, a primary mesenchyme lineage-specific cell surface protein in the sea urchin embryo

Development ◽  
1987 ◽  
Vol 101 (2) ◽  
pp. 255-265 ◽  
Author(s):  
J.A. Anstrom ◽  
J.E. Chin ◽  
D.S. Leaf ◽  
A.L. Parks ◽  
R.A. Raff
Keyword(s):  
Cell Surface ◽  
Sea Urchin ◽  
Sea Water ◽  
Surface Protein ◽  
Specific Cell ◽  
Cell Surface Protein ◽  
Sea Urchin Embryo ◽  
A Cell ◽  
Mesenchyme Cells ◽  
Primary Mesenchyme Cells

In this report, we use a monoclonal antibody (B2C2) and antibodies against a fusion protein (Leaf et al. 1987) to characterize msp130, a cell surface protein specific to the primary mesenchyme cells of the sea urchin embryo. This protein first appears on the surface of these cells upon ingression into the blastocoel. Immunoelectronmicroscopy shows that msp130 is present in the trans side of the Golgi apparatus and on the extracellular surface of primary mesenchyme cells. Four precursor proteins to msp130 are identified and we show that B2C2 recognizes only the mature form of msp130. We demonstrate that msp130 contains N-linked carbohydrate groups and that the B2C2 epitope is sensitive to endoglycosidase F digestion. Evidence that msp130 is apparently a sulphated glycoprotein is presented. The recognition of the B2C2 epitope of msp130 is disrupted when embryos are cultured in sulphate-free sea water. In addition, two-dimensional immunoblots show that msp130 is an acidic protein that becomes substantially less acidic in the absence of sulphate. We also show that two other independently derived monoclonal antibodies, IG8 (McClay et al. 1983; McClay, Matranga & Wessel, 1985) and 1223 (Carson et al. 1985), recognize msp130, and suggest this protein to be a major cell surface antigen of primary mesenchyme cells.

Autonomous and non-autonomous differentiation of ectoderm in different sea urchin species

Development ◽  
1995 ◽  
Vol 121 (5) ◽  
pp. 1497-1505 ◽  
Author(s):  
A.H. Wikramanayake ◽  
B.P. Brandhorst ◽  
W.H. Klein
Keyword(s):  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Protein A ◽  
Cellular Interactions ◽  
V Protein ◽  
Lytechinus Pictus ◽  
Sea Urchin Embryo ◽  
Oral Ectoderm ◽  
Temporal And Spatial

During early embryogenesis, the highly regulative sea urchin embryo relies extensively on cell-cell interactions for cellular specification. Here, the role of cellular interactions in the temporal and spatial expression of markers for oral and aboral ectoderm in Strongylocentrotus purpuratus and Lytechinus pictus was investigated. When pairs of mesomeres or animal caps, which are fated to give rise to ectoderm, were isolated and cultured they developed into ciliated embryoids that were morphologically polarized. In animal explants from S. purpuratus, the aboral ectoderm-specific Spec1 gene was activated at the same time as in control embryos and at relatively high levels. The Spec1 protein was restricted to the squamous epithelial cells in the embryoids suggesting that an oral-aboral axis formed and aboral ectoderm differentiation occurred correctly. However, the Ecto V protein, a marker for oral ectoderm differentiation, was detected throughout the embryoid and no stomodeum or ciliary band formed. These results indicated that animal explants from S. purpuratus were autonomous in their ability to form an oral-aboral axis and to differentiate aboral ectoderm, but other aspects of ectoderm differentiation require interaction with vegetal blastomeres. In contrast to S. purpuratus, aboral ectoderm-specific genes were not expressed in animal explants from L. pictus even though the resulting embryoids were morphologically very similar to those of S. purpuratus. Recombination of the explants with vegetal blastomeres or exposure to the vegetalizing agent LiCl restored activity of aboral ectoderm-specific genes, suggesting the requirement of a vegetal induction for differentiation of aboral ectoderm cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental analysis of the control of expression of the homeobox-gene Msx-1 in the developing limb and face

Development ◽  
1993 ◽  
Vol 119 (1) ◽  
pp. 41-48 ◽  
Author(s):  
J.M. Brown ◽  
S.E. Wedden ◽  
G.H. Millburn ◽  
L.G. Robson ◽  
R.E. Hill ◽  
...  
Keyword(s):  
Homeobox Gene ◽  
Host Tissue ◽  
Face To Face ◽  
Limb Mesenchyme ◽  
The Face ◽  
Mesenchyme Cells ◽  
Species Specific ◽  
Signalling Systems ◽  
Control Of Expression

Mouse mesenchyme was grafted into chick embryos to investigate the control of mesenchymal expression of Msx-1 in the developing limb and face. In situ hybridization, using species-specific probes, allows a comparison between Msx-1 expression in the graft and the host tissue. The results show that Msx-1 expression in both limb-to-limb and face-to-face grafts corresponds closely with the level of Msx-1 expression in the surrounding chick mesenchyme. Cells in grafts that end up within the host domain of Msx-1 express the gene irrespective of whether they were from normally expressing, or non-expressing, regions. Therefore Msx-1 expression in both the developing limb and the developing face appears to be position-dependent. Mesenchyme from each of the three major facial primordia behaved in the same way when grafted to the chick maxillary primordium. Reciprocal grafts between face and limb gave a different result: Msx-1 expression was activated when facial mesenchyme was grafted to the limb but not when limb mesenchyme was grafted to the face. This suggests either that there are quantitative or qualitative differences in two local signalling systems or that additional factors determine the responsiveness of the mesenchyme cells.

scholarly journals Encoding regulatory state boundaries in the pregastrular oral ectoderm of the sea urchin embryo

2014 ◽  
Vol 111 (10) ◽  
pp. E906-E913 ◽  
Author(s):  
Enhu Li ◽  
Miao Cui ◽  
Isabelle S. Peter ◽  
Eric H. Davidson
Keyword(s):  
Spatial Pattern ◽  
Sea Urchin ◽  
Regulatory Network ◽  
Regulatory State ◽  
Apical Plate ◽  
Sea Urchin Embryo ◽  
Oral Ectoderm ◽  
Positive Regulators ◽  
Gene Regulatory ◽  
Per Se

By gastrulation the ectodermal territories of the sea urchin embryo have developed an unexpectedly complex spatial pattern of sharply bounded regulatory states, organized orthogonally with respect to the animal/vegetal and oral/aboral axes of the embryo. Although much is known of the gene regulatory network (GRN) linkages that generate these regulatory states, the principles by which the boundaries between them are positioned and maintained have remained undiscovered. Here we determine the encoded genomic logic responsible for the boundaries of the oral aspect of the embryo that separate endoderm from ectoderm and ectoderm from neurogenic apical plate and that delineate the several further subdivisions into which the oral ectoderm per se is partitioned. Comprehensive regulatory state maps, including all spatially expressed oral ectoderm regulatory genes, were established. The circuitry at each boundary deploys specific repressors of regulatory states across the boundary, identified in this work, plus activation by broadly expressed positive regulators. These network linkages are integrated with previously established interactions on the oral/aboral axis to generate a GRN model encompassing the 2D organization of the regulatory state pattern in the pregastrular oral ectoderm of the embryo.

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