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W H Klein

Publications and source records attributed to W H Klein.

At least 55 records · Page 3Linked to original sources
PubMed1995

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

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)

Animals↗
PubMed1994

Multiple Otx binding sites required for expression of the Strongylocentrotus purpuratus Spec2a gene.

The control region of the aboral ectoderm-specific Spec2a gene of Strongylocentrotus purpuratus contains a 188-bp enhancer element, the RSR enhancer, required for temporal activation and aboral ectoderm/mesenchyme cell expression. Within the enhancer is a positive cis-regulatory element with the core consensus sequence TAATCC, which is capable of binding the sea urchin orthodenticle-related homeobox protein SpOtx. In this report, we extend our analysis of the RSR enhancer by dissecting it into smaller pieces and testing these pieces in an enhancer activation assay. The 188-bp enhancer region could not be divided without partial loss of activity, and two of the three pieces tested exhibited some activity. Using site-directed mutagenesis, we showed that three Otx consensus binding sites were responsible for the activity of the enhancer, acting in a non-cooperative manner to yield full activity. Mutagenizing the three Otx sites and a fourth one just upstream abolished all activity in the context of the complete Spec2a control region. Bandshift analysis revealed that the Otx sites were able to bind SpOtx, suggesting that this transcription factor mediates positive control at these sites. Non-SpOtx binding sites overlapping two of the Otx sites may also play a role in Spec2a expression. Using a lacZ reporter gene, we showed that a 76-bp DNA fragment containing two of the Otx sites was sufficient for aboral ectoderm/mesenchyme cell expression. These results suggest that the RSR enhancer plus an upstream DNA element required for mesenchyme cell repression are necessary and sufficient for the proper temporal activation and aboral ectoderm expression of the Spec2a gene and that the Otx elements play a positive role in this process.

Animals↗
PubMed1993

Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene.

Myogenin is a muscle-specific transcription factor that can induce myogenesis in a variety of cell types in tissue culture. To test myogenin's role in vivo, mice homozygous for a targeted mutation in the myogenin gene were generated. These mice survive fetal development but die immediately after birth and show a severe reduction of all skeletal muscle. Myogenin-mutant mice differ from mice carrying mutations in genes for the related myogenic factors Myf5 and MyoD, which have no muscle defects. Myogenin is therefore essential for the development of functional skeletal muscle.

Animals↗
PubMed1993

A positive cis-regulatory element with a bicoid target site lies within the sea urchin Spec2a enhancer.

Activation of the aboral ectoderm-specific Spec2a gene in blastula-stage sea urchin embryos requires an upstream regulatory region that is part of a repetitive sequence element (RSR) associated with all Spec1/Spec2 genes. Deletions from the 5' end of Spec2a-flanking DNA, monitored for activity using the sea urchin embryo gene-transfer expression system, indicated that this regulatory region has multiple DNA elements and that no positive element lies upstream of the RSR. We mapped the regulatory region using Spec2a fragments containing RSR sequences fused to an SV40 minimal promoter. The region between base pairs -443 and -631, defined as the RSR enhancer, was essential for maximal activity and conferred preferential aboral ectoderm expression to a lacZ reporter gene. Expression was not fully restricted to aboral ectoderm, however, suggesting that negative spatial elements are also associated with the proper activation of Spec2a. DNaseI footprinting and band-shift analysis of the RSR enhancer identified an A/T-rich DNA element, the A/T palindrome. This element binds a single 45-kDa nuclear protein, the A/T palindrome binding protein (A/TBP), whose specificity suggests a possible relationship with the bicoid-class homeodomain proteins. Mutations of the A/T palindrome are incapable of binding the 45-kDa protein and lower promoter activity by eight-fold. DNA-binding activity for A/TBP is low in unfertilized eggs, increases by the 16-cell stage and continues rising in blastulae. These data suggest that A/TBP plays a major role in the activation of the Spec2a gene in aboral ectoderm cells.

Animals↗
PubMed1993

Developmental potential of muscle cell progenitors and the myogenic factor SUM-1 in the sea urchin embryo.

During sea urchin development, esophageal muscle arises from secondary mesenchyme cells, descendants of the vegetal plate that delaminate from the coelomic epithelium at the end of gastrulation. In lithium-induced exogastrulae, where vegetal plate descendants evert rather than invaginate, myogenesis occurs normally, indicating that myocyte progenitors do not have to be near the future stomodeum for differentiation to occur. Vegetal plate descendants isolated along with the extracellular matrix at different times during gastrulation produce differentiated myocytes in culture as monitored by staining with a myosin heavy chain antibody. Vegetal isolates prepared at mid-gastrulation or later consistently produce differentiated myocytes whose form and position resembled their counterparts in the intact embryo, whereas vegetal isolates prepared a few hours earlier while capable of gut differentiation, as evidenced by the de novo synthesis of the endodermal surface marker Endo 1, did not produce differentiated myocytes. These results suggest that sometime after early gastrulation, a subset of secondary mesenchyme cells are competent to differentiate into muscle cells. RNase protection assays showed that the accumulation of sea urchin myogenic factor (SUM-1) mRNA is likely to be coincident with the earliest demonstrable commitment of myogenic precursors. Premature expression of SUM-1 coding sequences in mesenchyme blastulae resulted in the activation of muscle-specific enhancer elements, demonstrating that SUM-1 can function precociously in the early embryo. However, SUM-1 expressed in this manner did not activate the endogenous MHC gene, nor induce premature or ectopic production of muscle cells.

Amino Acid Sequence↗
PubMed1992

Members of the USF family of helix-loop-helix proteins bind DNA as homo- as well as heterodimers.

We have isolated human cDNA clones for USF2, a new member of the upstream stimulatory factor (USF) family of transcription factors. Analysis of these clones revealed the existence of highly conserved elements in the C terminal region of all USF proteins. These include the basic region, helix-loop-helix (HLH) motif, and, in the case of the human proteins, the C-terminal leucine repeat (LR). In addition, a highly conserved USF-specific domain is located immediately upstream of the basic region. Using in vitro translated proteins, we found that all members of the USF family bound DNA as dimers. The N-terminal portion of USF, including the USF-specific domain, was entirely dispensable for dimer formation and DNA-binding. However, deletion mutants of USF2 lacking the LR were deficient in DNA-binding activity. Interestingly, each of the USF proteins could form functional heterodimers with the other family members, including the sea urchin USF, which does not have a LR motif. This indicates that the conserved LR in human USF is not required for dimer formation, and influences only indirectly DNA-binding.

Amino Acid Sequence↗
PubMed1991

A myogenic factor from sea urchin embryos capable of programming muscle differentiation in mammalian cells.

Using the basic helix-loop-helix domain of the myogenic factor myogenin as a probe, we identified a clone from a sea urchin cDNA library with considerable sequence similarity to the vertebrate myogenic factors. This cDNA, sea urchin myogenic factor 1 (SUM-1), transactivated a muscle creatine kinase-chloramphenicol acetyltransferase reporter gene in 10T1/2 fibroblasts to a level comparable to that of the vertebrate myogenic factors. In addition, bacterially expressed beta-galactosidase-SUM-1 fusion protein interacted directly with the kappa E-2 site in the muscle creatine kinase enhancer core as assayed by electrophoretic mobility shift assays. Stably transfected SUM-1 activated the muscle differentiation program and converted 10T1/2 cells from fibroblasts to myotubes. In sea urchin embryos, SUM-1 RNA was not detected before gastrulation. It accumulated to its highest levels during the prism stage when myoblasts were first detected by myosin immunostaining and then diminished as myocytes differentiated. SUM-1 protein was localized in secondary mesenchyme cells when they could first be identified as muscle cells by myosin immunostaining. These results implicate SUM-1 as a regulatory factor involved in the early decision of a pluripotent secondary mesenchyme cell to convert to a myogenic fate. SUM-1 is an example of an invertebrate myogenic factor that is capable of functioning in mammalian cells.

Amino Acid Sequence↗
PubMed1991

Structure and promoter activity of the LpS1 genes of Lytechinus pictus. Duplicated exons account for LpS1 proteins with eight calcium binding domains.

The LpS1 genes of the sea urchin Lytechinus pictus are activated early in development in aboral ectoderm cells. They therefore have ontogenic properties similar to their counterparts in Stronglyocentrotus purpuratus, the Spec genes. Both gene families encode proteins belonging to the calmodulin superfamily as evidenced by the presence of distinct EF-hand (helix-loop-helix) domains. The presence of eight EF-hand domains in LpS1 proteins suggests that the LpS1 genes arose from a duplication of an ancestral Spec-like gene. The LpS1 genes were further analyzed to increase our understanding of the mechanisms underlying their evolution and activation in aboral ectoderm cells. Genomic DNA blot analysis showed two LpS1 genes, LpS1 alpha and LpS1 beta, which did not appear to be closely linked. LpS1 genomic clones were isolated by screening an L. pictus genomic library with an LpS1 cDNA clone, and partial gene structures for both LpS1 alpha and LpS1 beta were constructed. These revealed internal duplication of the LpS1 genes that accounted for the eight EF-hand domains in the LpS1 proteins. Duplication of exon 1 in both genes suggested four different LpS1 proteins could be derived from the LpS1 genes. Primer extension to map the transcriptional initiation sites of the LpS1 genes and sequencing analysis showed there was little in common among the 5'-flanking regions of the LpS1 and Spec genes except for the presence of a binding site for the transcription factor USF. A sea urchin gene-transfer expression system showed that 762 base pairs (bp) of 5'-flanking DNA and 17 bp of 5'-untranslated leader sequence of the LpS1 beta gene were sufficient for correct temporal and spatial expression of reporter chloramphenicol acetyltransferase and lacZ genes in sea urchin embryos. Deletions at the 5' end to either 511 or 368 bp resulted in a 3-4 fold decrease in chloramphenicol acetyltransferase activity and disrupted the exclusive activation of the lacZ gene in aboral ectodermal cells. Based on a lineage analysis among the LpS1 and Spec gene families and other related genes, we propose a model in which LpS1 genes evolved from a series of duplications of an ancestral Spec-like gene.

Amino Acid Sequence↗
PubMed1991

Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1.

The zinc-finger transcription factor GATA-1 (previously known as GF-1, NF-E1 or Eryf 1 binds to GATA consensus elements in regulatory regions of the alpha- and beta-globin gene clusters and other erythroid cell-specific genes. Analysis of the effects of mutations in GATA-binding sites in cell culture and in binding assays in vitro, as well as transactivation studies with GATA-1 expression vectors in heterologous cells, have provided indirect evidence that this factor is involved in the activation of globin and other genes during erythroid cell maturation. GATA-1 is also expressed in megakaryocytes and mast cells, but not in other blood cell lineages or in non-haemopoietic cells. To investigate the role of this factor in haematopoiesis in vivo, we disrupted the X-linked GATA-1 gene by homologous recombination in a male (XY) murine embryonic stem cell line and tested the GATA-1-deficient cells for their ability to contribute to different tissues in chimaeric mice. The mutant embryonic stem cells contributed to all non-haemopoietic tissues tested and to a white blood cell fraction, but failed to give rise to mature red blood cells. This demonstrates that GATA-1 is required for the normal differentiation of erythroid cells, and that other GATA-binding proteins cannot compensate for its absence.

Animals↗
PubMed1991

Sea urchin USF: a helix-loop-helix protein active in embryonic ectoderm cells.

We previously characterized a DNA-binding factor in nuclear extracts of Strongylocentrotus purpuratus embryos that bound Spec gene promoters, was ectoderm specific, and had properties similar to the vertebrate transcription factor USF. Here we describe a cDNA clone, suUSF, isolated from an S. purpuratus cDNA library, with sequence homology to human USF. Spec gene promoter fragments formed sequence-specific complexes with suUSF, and antibodies against suUSF inhibited binding activity in nuclear extracts. Reaction of USF-site containing probes with filter-bound nuclear proteins demonstrated that suUSF binding activity was enriched in ectoderm cells, and immunoblotting showed a similar ectoderm enrichment. These data demonstrated that suUSF was responsible for the ectoderm-specific activity observed in sea urchin extracts.

Amino Acid Sequence↗
PubMed1991

A G-string positive cis-regulatory element in the LpS1 promoter binds two distinct nuclear factors distributed non-uniformly in Lytechinus pictus embryos.

The LpS1 alpha and beta genes of Lytechinus pictus are activated at the late cleavage stage of embryogenesis, with LpS1 mRNAs accumulating only in lineages contributing to aboral ectoderm. We had shown previously that 762 bp of 5' flanking DNA from the LpS1 beta gene was sufficient for proper temporal and aboral ectoderm specific expression. In the present study, we identified a strong positive cis-regulatory element at -70 bp to -75 bp in the LpS1 beta promoter with the sequence (G)6 and a similar, more distal cis-element at -721 bp to -726 bp. The proximal 'G-string' element interacted with two nuclear factors, one specific to ectoderm and one to endoderm/mesoderm nuclear extracts, whereas the distal G-string element interacted only with the ectoderm factor. The ectoderm and endoderm/mesoderm G-string factors were distinct based on their migratory behavior in electrophoretic mobility shift assays, binding site specificities, salt optima and EDTA sensitivity. The proximal G-string element shared homology with a binding site for the mammalian transcription factor IF1, a protein that binds to negative cis-regulatory elements in the mouse alpha 1(I) and alpha 2(I) collagen gene promoters. Competition experiments using wild-type and mutant oligonucleotides indicated that the ectoderm G-string factor and IF1 have similar recognition sites. Partially purified IF1 specifically bound to an oligonucleotide containing the proximal G-string of LpS1 beta. From our results, we suggest that the ectoderm G-string factor, a member of the G-rich DNA-binding protein family, activates the LpS1 gene in aboral ectoderm cells by binding to the LpS1 promoter at the proximal G-string site.

Animals↗
PubMed1990

Temporal and spatial transcriptional regulation of the aboral ectoderm-specific Spec genes during sea urchin embryogenesis.

mRNAs for Spec 1 and Spec 2 of Strongylocentrotus purpuratus and LpS1 of Lytechinus pictus accumulate only in the aboral ectoderm of developing embryos. In vitro nuclear transcription assays were done to study the transcriptional regulation of these cell type-specific genes. Spec 1, Spec 2c, and Spec 2d genes all appeared to be transcriptionally activated at the late cleavage-early blastula stage of S. purpuratus. Differences in the relative transcription rates during development appeared to play a major role in determining the relative levels of the various Spec mRNAs. The L. pictus LpS1 gene was transcriptionally activated at a similar developmental time as the corresponding S. purpuratus genes. Nuclei from gastrula or pluteus ectodermal and endodermal/mesodermal cell fractions were used to demonstrate that Spec 1 and LpS1 genes were transcriptionally active in ectoderm nuclei but not in endoderm/mesoderm nuclei, suggesting that in vivo the Spec 1 and LpS1 genes are spatially controlled at the transcriptional level. Estimations of the absolute rate constants for Spec 1 transcription were made at the late cleavage, mesenchyme blastula, and midgastrula stages. Calculations using these rate constants and the known levels of Spec 1 mRNA suggested that Spec 1 mRNA stability gradually increased throughout development.

Animals↗
PubMed1990

Myosin heavy chain accumulates in dissimilar cell types of the macromere lineage in the sea urchin embryo.

In this study, myosin heavy chain from sea urchin pluteus larvae was characterized by analysis of a 2.5-kb cDNA clone. DNA sequence of 1465 bp demonstrated a 71% similarity in the deduced amino acid sequence to the embryonic rat skeletal muscle sequence. Antibodies generated against a polypeptide encoded by the open reading frame of the cDNA clone specifically identified a 210-kDa myosin protein which accumulated in 8-12 muscle cells differentiating bilateral to the esophagus beginning at early larval stages. This same myosin also accumulated in cells of the endodermal epithelium that comprise the three sphincters of the larval gut. Thus, a gene encoding myosin heavy chain is expressed in dissimilar cell types of the macromere lineage, and the pattern of accumulation in the gut identifies functionally distinct cells of the endodermal epithelium.

Amino Acid Sequence↗
PubMed1990

Repetitive DNA sequences linked to the sea urchin spec genes contain transcriptional enhancer-like elements.

The 5' flanking DNA of three related Strongylocentrotus purpuratus genes, Spec1, Spec2a, and Spec2c, were analyzed with respect to structure and cis-regulatory activity. The structural features of DNA sequences upstream of the first intron were highly unusual and implicated certain regions as sites of coordinate control for gene expression. By aligning the genes with a common upstream 600-bp repetitive DNA sequence element, termed RSR, it was shown that a conserved DNA block of approximately 800 bp extended from the 3' end of the first exon to the 5' end of the RSR element. In Spec2a, the conserved sequence block was a continuous stretch of DNA, but in Spec1 and Spec2c, 2.5 to 3 kb of inserted DNA bounded by short direct repeats interrupted the conserved sequence block, thus changing the relative placement of the RSR element and other 5' flanking DNA. Deletion of XhoI fragments containing the 5' half but not the 3' half of the RSR element resulted in a significant decrease in chloroamphenicolacetyl transferase (CAT) activity when Spec-CAT reporter gene fusion plasmids were injected into Lytechinus pictus eggs. These results strongly suggested, but did not prove, that the sequences held in common among the XhoI fragments, that is, the 5' half of the RSR elements, were responsible for the decrease in CAT activity. The Spec2a gene was particularly sensitive to deletions of the XhoI fragment containing the 5' half of the RSR element. The deleted element had several enhancer-like properties when inserted back into various test plasmids: it could be positioned in locations different from the transcriptional start site; in some but not all cases, it could be made to work in the reverse orientation; and it could drive expression of the CAT gene using an SV40 promoter or cryptic promoter elements. These findings suggested that an enhancer-like element important for Spec gene expression was contained within a repetitive DNA sequence. Genomic DNA blots suggested that there are many more of these RSR elements than there are Spec genes.

Animals↗
PubMed1990

Regulatory elements from the related spec genes of Strongylocentrotus purpuratus yield different spatial patterns with a lacZ reporter gene.

The Spec1 and Spec2 genes of Strongylocentrotus purpuratus are closely associated with the differentiation of aboral ectoderm. To examine cis-regulatory elements involved in the spatial expression of the Spec genes, we fused the Escherichia coli lacZ gene containing a nuclear targeting signal to 5'flanking DNA plus 5' untranslated leader sequences from Spec1, Spec2a, and Spec2c. All three genes contain 700 bp of highly conserved DNA in their upstream regions, but in Spec1 and Spec2c large insertions interrupt the conserved regions. The Spec-lacZ reporter gene plasmids were microinjected into eggs of S. purpuratus, Lytechinus variegatus, and L. pictus, and beta-galactosidase activity was determined in situ by X-gal staining. The Spec2a-lacZ fusion gene, which contained 1516 bp of 5' flanking DNA and 18 bp of 5' untranslated leader sequence, was preferentially expressed in aboral ectoderm cells in all three species. The Spec1-lacZ fusion gene was expressed in a strikingly different fashion--preferentially in primary and secondary mesenchyme cells, occasionally in aboral ectoderm cells, and less often in oral ectoderm and endoderm cells. The staining pattern was the same in either homologous or heterologous embryos. The Spec2c-lacZ fusion gene, like Spec2a-lacZ, was preferentially expressed in aboral ectoderm, but staining of other cell types was frequently observed. To further delineate sequences required for correct spatial expression, we deleted 800 bp of 5' flanking DNA from the Spec2a-lacZ fusion gene, resulting in a delta Spec2a-lacZ fusion gene that contained only the conserved DNA region. This gene fusion showed preferential expression in aboral ectoderm cells. However, the cell type specificity was not as great as with the parental Spec2a-lacZ plasmid. These experiments implied that the conserved DNA region, associated with all Spec genes examined, was insufficient for complete aboral ectoderm specificity, and suggested that a spatial repressor element existed between -1516 and -697 bp in the 5' flanking DNA of Spec2a.

Animals↗
PubMed1990

Localization of the sea urchin Spec3 protein to cilia and Golgi complexes of embryonic ectoderm cells.

Expression of the Spec3 gene of Strongylocentrotus purpuratus is associated with ectodermal ciliogenesis. An antiserum was raised against the amino terminus of the deduced Spec3 amino acid sequence and used for immunofluorescent staining. Cilia and an apical structure at the base of the stained cilium of each ectodermal cell stained intensely in gastrula and later stage embryos. Microtubule-depolymerizing agents dispersed the concentrated spot of apical staining, suggesting a localization of Spec3 antigen to the Golgi complex. Immunogold electron microscopy confirmed the localization of Spec3 antigen on cilia and in the Golgi complex. Spec3 antigen showed a diffuse punctate staining pattern in the ectodermal cytoplasm of hatching blastula when Spec3 transcripts are most prevalent, suggesting that after synthesis, Spec3 is sequestered in the Golgi complex before appearing on cilia. Whereas the predicted Mr of the Spec3 protein is 21,600, immunoblotting with S. purpuratus proteins indicated that a Spec3 antigen was concentrated in cilia and migrated as an SDS-resistant aggregate of Mr approximately 350,000. Spec3 is also concentrated in cilia of Lytechinus pictus but the protein migrated with an Mr approximately 23,000 in this species. The S. purpuratus Spec3 antigen remains associated with the ciliary axoneme after extraction of membrane proteins.

Amino Acid Sequence↗
PubMed1990

Ectoderm nuclei from sea urchin embryos contain a Spec-DNA binding protein similar to the vertebrate transcription factor USF.

The Spec gene family of Stronglyocentrotus purpuratus is expressed exclusively in aboral ectoderm cells during embryogenesis. To investigate the regulation of Spec gene activity, the region around the Spec1 transcriptional initiation site was analyzed for sites of protein-DNA interaction. One high-affinity site bound a factor termed SpF1 within the Spec1 5' untranslated leader region at position +39 to +60. The core sequence recognized by SpF1, CACGTG, is the same as that of the upstream stimulatory factor (USF), a widely occurring vertebrate transcription factor containing a myc-HLH motif. A comparison of USF- and SpF1-binding activities suggested that SpF1 was a sea urchin version of USF. SpF1 activity was detectable only in ectoderm cells of the embryo, implying that it has a role as a cell type-specific transcription factor. SpF1-binding sites were also found upstream of the Spec2a and Spec2c genes in the same conserved sequence block as Spec1. Extracts from Lytechinus pictus embryos showed an SpF1-like activity, suggesting that SpF1 is conserved in sea urchins. Surprisingly, changes in the Spec1, Spec2a, or Spec2c genes that removed or modified the SpF1-binding site had no effect on expression when reporter gene fusions containing these mutations were injected into sea urchin eggs and analyzed for expression during embryogenesis. We propose that, while SpF1 may not be essential for expression of the exogenously introduced reporter genes, it may be required for proper regulation of the endogenous Spec genes.

Animals↗