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B P Brandhorst

Publications and source records attributed to B P Brandhorst.

At least 19 recordsLinked to original sources
PubMed2006

A genomic view of the sea urchin nervous system.

The sequencing of the Strongylocentrotus purpuratus genome provides a unique opportunity to investigate the function and evolution of neural genes. The neurobiology of sea urchins is of particular interest because they have a close phylogenetic relationship with chordates, yet a distinctive pentaradiate body plan and unusual neural organization. Orthologues of transcription factors that regulate neurogenesis in other animals have been identified and several are expressed in neurogenic domains before gastrulation indicating that they may operate near the top of a conserved neural gene regulatory network. A family of genes encoding voltage-gated ion channels is present but, surprisingly, genes encoding gap junction proteins (connexins and pannexins) appear to be absent. Genes required for synapse formation and function have been identified and genes for synthesis and transport of neurotransmitters are present. There is a large family of G-protein-coupled receptors, including 874 rhodopsin-type receptors, 28 metabotropic glutamate-like receptors and a remarkably expanded group of 161 secretin receptor-like proteins. Absence of cannabinoid, lysophospholipid and melanocortin receptors indicates that this group may be unique to chordates. There are at least 37 putative G-protein-coupled peptide receptors and precursors for several neuropeptides and peptide hormones have been identified, including SALMFamides, NGFFFamide, a vasotocin-like peptide, glycoprotein hormones and insulin/insulin-like growth factors. Identification of a neurotrophin-like gene and Trk receptor in sea urchin indicates that this neural signaling system is not unique to chordates. Several hundred chemoreceptor genes have been predicted using several approaches, a number similar to that for other animals. Intriguingly, genes encoding homologues of rhodopsin, Pax6 and several other key mammalian retinal transcription factors are expressed in tube feet, suggesting tube feet function as photosensory organs. Analysis of the sea urchin genome presents a unique perspective on the evolutionary history of deuterostome nervous systems and reveals new approaches to investigate the development and neurobiology of sea urchins.

Animals↗
PubMed2001

Regulation of metamorphosis in ascidians involves NO/cGMP signaling and HSP90.

Treatment of larvae of the ascidians Boltenia villosa (Family: Pyuridae) and Cnemidocarpa finmarkiensis (Family: Styelidae) with drugs that inhibit the function of the molecular chaperone HSP90 increased the frequency of tail resorption, the primary morphogenetic event of metamorphosis. If treatment was initiated at hatching, metamorphic events subsequent to tail resorption failed to occur, indicating an ongoing role for HSP90 during morphogenesis. Removal of tails from heads of mature, but not newly hatched larvae, induced metamorphosis of the head. Decapitation experiments indicate that the capacity of tails to shorten in response to inhibition of HSP90 function requires communication with heads. To identify candidate proteins with which HSP90 may interact to regulate metamorphosis, we noted that in mammalian cells, nitric oxide synthase (NOS) interacts with HSP90 and its activity is sensitive to drugs that inhibit HSP90 function. In addition, nitric oxide (NO) signaling in the marine snail Ilyanassa obsoleta is an important regulator of metamorphosis. Inhibition of NOS activity in these ascidian larvae with L-NAME increased the frequency of metamorphosis, consistent with a putative interaction of NOS and HSP90. NOS is present in tail muscle cells, implicating them as targets for the drug treatments, consistent with the decapitation experiments. Inhibition of soluble guanylyl cyclase, the most common effector of NO signaling, also increased the frequency of metamorphosis. In contrast to treatment with anti-HSP90 drugs, metamorphosis induced with L-NAME or ODQ was complete. The results presented suggest that an HSP90-dependent, NO-based regulatory mechanism localized in tails represses ascidian metamorphosis. We discuss these results in relation to the induction of ascidian metamorphosis by several unrelated agents.

Animals↗
PubMed2001

Skeletogenesis in sea urchin interordinal hybrid embryos.

Reciprocal interordinal crosses were made between the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus. Previous research indicated that the expression of many L. pictus genes is reduced in the hybrid embryos. The S. purpuratus gene encoding the spicule matrix protein SM50 and the L. pictus gene encoding its orthologue LSM34 were both expressed at normal levels per gene copy in hybrid embryos, and in about 32 skeletogenic primary mesenchyme cells (PMCs) in hybrid and natural gastrulae. In many embryos of all crosses, 16 PMCs initially ingressed, while 32-64 PMCs were present in gastrulae. The skeletal spicules of most hybrid plutei were predominantly like those of S. purpuratus, consistent with the predominance of expression of S. purpuratus genes in hybrid embryos. The spicules of some hybrid plutei showed features characteristic of L. pictus, such as recurrent rods, branched body rod tips, or convergent ventral transverse rods; a few hybrid spicules were predominantly like those of L. pictus. Based on our observations and the literature, we propose the following. Cues from the ectodermal epithelium position the PMCs as they elaborate the initial triradiate spicules. Their orientation and outgrowth appears to be responsible for the convergence of the tips of body rods in most S. purpuratus and hybrid embryos, unlike in most L. pictus embryos. Variations among hybrid and natural embryos in skeletal branching pattern reflect differences in interpretation by PMCs of patterning cues produced by the ectodermal epithelium that probably have similar spatial distributions in the two species.

Animals↗
PubMed2001

NO/cGMP signaling and HSP90 activity represses metamorphosis in the sea urchin Lytechinus pictus.

Nitric oxide (NO) signaling repressively regulates metamorphosis in two solitary ascidians and a gastropod. We present evidence for a similar role in the sea urchin Lytechinus pictus. NO commonly signals via soluble guanylyl cyclase (sGC). Nitric oxide synthase (NOS) activity in some mammalian cells, including neurons, depends on the molecular chaperone heat shock protein 90 (HSP90); this may be so in echinoid larvae as well. Pluteus larvae containing juvenile rudiments were treated with either radicicol L- or D-nitroarginine-methyl-ester (L-NAME and D-NAME), or IH-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), inhibitors of HSP90, NOS, and sGC, respectively. In all instances, drug treatment significantly increased the frequency of metamorphosis. SNAP, a NO donor, suppressed the inductive properties of L-NAME and biofilm, a natural inducer of metamorphosis. NADPH diaphorase histochemistry indicated NOS activity in cells in the lower lip of the larval mouth, the preoral hood, the gut, and in the tube feet of the echinus rudiment. Histochemical staining coincided with NOS immunostaining. Microsurgical removal of the oral hood or the pre-oral hood did not induce metamorphosis, but larvae lacking these structures retained the capacity to metamorphose in response to ODQ. We propose that the production of NO repressively regulates the initiation of metamorphosis and that a sensory response to environmental cues reduces the production of NO, and consequently cGMP, to initiate metamorphosis.

Animals↗
PubMed1997

Metallothionein gene expression in embryos of the sea urchin Lytechinus pictus.

The metallothionein (MT) gene LpMT1 of the sea urchin Lytechinus pictus was characterized. The primary transcript of 3042 nucleotides includes four exons, as uniquely observed for other sea urchin MT genes, which are spliced to form a messenger RNA of 605 nucleotides. The deduced LpMT1 protein sequence includes 69 amino acids, more than observed for other MT proteins. For a high level of inducible activity, the LpMT1 promoter requires sequence elements in addition to the canonical regulatory elements identified for mammalian MT promoters. The promoter of the closely related LpMT2 gene is very active in spite of its lack of a distinctive poly(C) element included in a sequence tract required for fully induced activity of the LpMT1 promoter. In contrast to embryos of the sea urchin S. purpuratus in which MT mRNAs are restricted to the aboral ectoderm of uninduced embryos, no spatially preferential accumulation of MT mRNAs in L. pictus embryos was observed. The cisacting regulatory elements required for MT gene activity and the spatial specificity of MT gene expression in sea urchin embryos are considered. The LpMT1 and LpMT2 promoters constitute promiscuous promoters that can be induced to a high level of activity.

Amino Acid Sequence↗
PubMed1997

Efficient detection of DNA polymorphisms by fluorescent RAPD analysis.

A method is presented for the analysis of fluorescently labeled random amplified polymorphic DNA (FRAPD) fragments. A DNA sequencer and collection and analysis software were used to estimate the sizes of DNA fragments based on their mobilities relative to in-lane size markers. This allowed confident identification and comparison of FRAPD markers both within and between polyacrylamide gels. In comparison with analysis of RAPD products using ethidium bromide-stained agarose gels, fluorescent analysis improved the sensitivity, resolution and precision of sizing of RAPD products of about 50-2100 bp. FRAPD fragments produced from amplification of zebrafish DNA are informative as genetic markers that segregate with Mendelian inheritance. FRAPD analysis was found to be very efficient for identifying new DNA polymorphisms.

Animals↗
PubMed1996

Expression of the actin gene family in embryos of the sea urchin Lytechinus pictus.

The genome of the sea urchin Lytechinus pictus includes genes encoding four cytoskeletal actins LpC1-4 and the muscle actin LpM. Gene-specific probes corresponding to 3' noncoding termini have been used to characterize their patterns of expression by in situ hybridization. The gene encoding LpC1 actin, which is most similar in sequence to the Strongylocentrotus purpuratus CyI actin, has a complex developmental profile of expression. LpC1 transcripts become prominent in the archenteron and secondary mesenchyme cells of embryos, as well as in adult testis and ovary. The LpC2 actin gene is predominantly expressed in aboral ectoderm of embryos, similarly to the pattern of expression of its closest relatives in S. purpuratus, the SpCyIIIa and SpCyIIIb actin genes. The LpC3 actin gene is expressed at low levels in secondary mesenchyme cells. The LpC4 actin gene is expressed in a subset of primary mesenchyme cells which may be actively engaged in skeletogenesis. Transcripts of the LpM gene accumulate in esophageal muscle cells beginning during gastrulation before overt differentiation. Each of the L. pictus actin genes has a distinct pattern of expression, none of which is identical to that of any S. purpuratus actin gene. These results indicate that the regulation of expression of members of the actin gene family, even those likely to have common ancestors, has diverged as these sea urchin species diverged.

Actins↗
PubMed1996

Production of androgenetic zebrafish (Danio rerio).

To help investigate the evolutionary origin of the imprinting (parent-of-origin mono-allelic expression) of paternal genes observed in mammals, we constructed haploid and diploid androgenetic zebrafish (Danio rerio). Haploid androgenotes were produced by fertilizing eggs that had been X-ray irradiated to eliminate the maternal genome. Subsequent inhibition of the first mitotic division of haploid androgenotes by heat shock produced diploid androgenotes. The lack of inheritance of maternal-specific DNA markers (RAPD and SSR) by putative diploid and haploid androgenotes confirmed the androgenetic origin of their genomes. Marker analysis was performed on 18 putative androgenotes (five diploids and 13 haploids) from six families. None of 157 maternal-specific RAPD markers analyzed, some of which were apparently homozygous, were passed on to any of these putative androgenotes. A mean of 7.7 maternal-specific markers were assessed per family. The survival of androgenetic zebrafish suggests that if paternal imprinting occurs in zebrafish, it does not result in essential genes being inactivated when their expression is required for development. Production of haploid androgenotes can be used to determine the meiotic recombination rate in male zebrafish. Androgenesis may also provide useful information about the mechanism of sex determination in zebrafish.

Animals↗
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

Evolution of actin gene families of sea urchins.

The actin gene family of the sea urchin Lytechinus pictus includes a single muscle actin gene, LpM, and four cytoskeletal actin genes: LpC1, LpC2, LpC3, and LpC4. The origin and relationship of these actin genes to members of the actin gene family of the sea urchin Strongylocentrotus purpuratus were considered. Comparison of deduced amino acid sequences suggested a close relationship between LpC1 and the CyI-CyII subfamily of S. purpuratus actin genes, and between LpC2 and the CyIII subfamily of S. purpuratus actin genes; the muscle actin genes were orthologous. It is proposed that two divergent cytoskeletal actin genes of the common ancestral sea urchin gave rise by duplication to the extant cytoskeletal actin genes of these species, some of which have changed 3' noncoding sequences while others have maintained a terminus highly conserved among sea urchin actin genes.

Actins↗
PubMed1992

Mutually exclusive expression of the Strongylocentrotus purpuratus Spec1 gene and its Lytechinus pictus homologue in cells of hybrid embryos.

The expression of the Spec1 gene of Strongylocentrotus purpuratus and its Lytechinus pictus homologue LpS1 was analyzed in reciprocal hybrid embryos of these two species of sea urchin. While the time course of accumulation of Spec1 mRNA was nearly normal in hybrid embryo populations, the accumulation of LpS1 mRNA was not. This was particularly evident in plutei, where the level of LpS1 mRNA was less than 5% that in normal L. pictus plutei. In situ hybridization analysis of serial sections indicated that LpS1 mRNA was detectable in only about 2% of hybrid plutei in either cross, whereas Spec1 mRNA was present in nearly all hybrid plutei; expression of either homologue was appropriately restricted to the aboral ectoderm. In crosses of L. pictus eggs with S. purpuratus sperm (LpSp), about 1% of hybrid plutei expressed LpS1 RNA in most or all aboral ectoderm cells at normal levels, and did not express Spec1 RNA; in another 1% of the LpSp hybrid plutei the Spec1 and LpS1 transcripts were present at normal levels in complementary, non-overlapping patches of contiguous aboral ectoderm cells. In the reciprocal SpLp cross, each hybrid pluteus expressed either only the LpS1 gene (about 2%) or only the Spec1 gene throughout the aboral ectoderm. In SpLp hybrid gastrulae the level of LpS1 mRNA was less restricted; about 2% of the embryos contained only LpS1 RNA, and about half expressed only Spec1 transcripts, but in the remaining embryos Spec1 and LpS1 transcripts were coexpressed in the same aboral ectoderm cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Structure and expression of the polyubiquitin gene in sea urchin embryos.

A cloned Lytechinus pictus cDNA has been identified, which includes seven direct repeats of a 228 bp sequence encoding ubiquitin and about 450 bp of 3' noncoding sequence. The deduced amino acid sequence is identical to that of ubiquitins of other animals (though repeats 3 and 5 each have single amino acid substitutions at different positions). Southern blot analysis revealed that the sea urchin genome contains a single copy of the polyubiquitin gene, and the number of 228 bp repeat units appears to vary from seven to ten among different alleles; no other ubiquitin coding sequences were detected. The size distribution of polyubiquitin mRNA is polymorphic among different individuals, probably corresponding to the differences in copy number of the repetitive coding sequence. The abundance of cytoplasmic polyubiquitin RNA is constant throughout embryogenesis and is similar in ectoderm, endoderm, and mesoderm cells. The constant prevalence of polyubiquitin mRNA apparently results from a balance between ontogenetic changes in its rate of synthesis and its stability in the presence of actinomycin D. Accumulation of polyubiquitin RNA was not heat shock-inducible during embryogenesis.

Amino Acid Sequence↗
PubMed1991

Restricted expression of the Lytechinus pictus Spec1 gene homologue in reciprocal hybrid embryos with Strongylocentrotus purpuratus.

Hybrid embryos were derived from reciprocal crosses of Strongylocentrotus purpuratus and Lytechinus pictus sea urchins. The expression of proteins specific for L. pictus was restricted in these hybrid embryos, while this was not so for most proteins specific for S. purpuratus. In particular, the aboral ectoderm-specific calcium-binding protein Spec1 was expressed at normal levels in hybrid embryos, but its L. pictus homologue, LpS1, was considerably reduced. LpS1 mRNA accumulated in hybrid plutei to only 4-5% of its normal level. Transcription of the LpS1 gene was substantially reduced in hybrid embryos, as determined by a nuclear RNA run-on assay. Southern blot analysis of genomic DNA indicated that there was no detectable loss or rearrangement of LpS1 DNA in hybrid embryos. Thus, the Spec1 gene is expressed normally in hybrid embryos, but the transcription of its homologue, the LpS1 gene, is considerably restricted.

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↗
PubMed1988

Tandem duplication and divergence of a sea urchin protein belonging to the troponin C superfamily.

The Spec1 and Spec2 proteins of the sea urchin Strongylocentrotus purpuratus are related to calmodulin, troponin C, and myosin light chains by sequence similarity in their four calcium binding domains. These domains, the EF-hands, are distinct helix-loop-helix structures of about 40 amino acids. The Spec1 and Spec2 genes are expressed specifically in aboral ectoderm cells of the developing embryo; however, the function of the Spec proteins in these cells is unknown. To find conserved regions of the proteins that might be important for structure and function, Spec homologues from Lytechinus pictus, a distantly related sea urchin, were sought. L. pictus embryos do not synthesize detectable amounts of the 14,000-17,000-Da Spec proteins as determined by two-dimensional gel electro-phoresis, but do synthesize three 34,000-Da proteins that cross-react with Spec1 antibodies and display a similar ontogenetic pattern of expression. cDNA clones were isolated by hybridization to a synthetic oligonucleotide corresponding to the EF-hand. One clone, LpS1, encodes an mRNA with developmental properties like those of the S. purpuratus Spec mRNAs. However, LpS1 contains an open reading frame for a protein of 34,000 Da rather than 17,000 Da, and antibodies raised against part of the LpS1 reading frame demonstrate that LpS1 encodes a 34,000-Da protein in L. pictus embryos. The sequence of LpS1 reveals the presence of eight EF-hand domains, which share structural homology with the Spec1 or Spec2 EF-hands; however, little else in the protein sequence is conserved. The results support the hypothesis that the LpS1 gene arose from a duplication of an ancestral Spec gene and that the overall structural features of the Spec family of proteins are more conserved than the amino acid sequences.

Amino Acid Sequence↗
PubMed1988

Microtubule formation from maternal tubulins during sea urchin embryogenesis: measurement of soluble and insoluble tubulin pools.

The mass of tubulin protein in developing embryos of the sea urchin Lytechinus pictus was measured using a radiodilution immunoassay based on densitometric analysis of immunoprecipitated tubulins resolved electrophoretically. The tubulins constitute an average of 360 +/- 35 pg per egg, or 0.66% of the total protein, and there is no significant change in their concentration during embryogenesis. The masses of soluble and polymerized tubulin were measured for extracts prepared under conditions that stabilize microtubules. In eggs, a maximum of 14% of the tubulin is insoluble, and this increases throughout embryogenesis to 67% at pluteus stage (72 hr). The concentration of tubulin in eggs is at least 500 micrograms/ml, well above the critical concentration for tubulin assembly in vitro, yet microtubules have not been observed in eggs. The mass of newly synthesized tubulin, estimated from the mass of tubulin mRNA per embryo, accounts for a small fraction of the total tubulin by the end of gastrulation but for over half of the tubulin by the 72-hr pluteus stage. These observations are consistent with a model in which the declining level of unpolymerized tubulin controls the stability of tubulin mRNa, providing an autogenous regulation of the ontogenetic pattern of tubulin synthesis during sea urchin embryogenesis (Gong and Brandhorst, Development 102: 31-43).

Animals↗