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PUBLICATIONS
2011 Acoelomorph flatworms are deuterostomes related to Xenoturbella Hervé Philippe,Henner Brinkmann, Richard R. Copley, Leonid L. Moroz, Hiroaki Nakano, Albert J. Poustka, Andreas Wallberg, Kevin J. Peterson & Maximilian J. Telford Xenoturbellida and Acoelomorpha
are marine worms with contentious ancestry. Both were originally
associated with the flatworms (Platyhelminthes), but molecular data
have revised their phylogenetic positions, generally linking
Xenoturbellida to the deuterostomes and positioning the
Acoelomorpha as the most basally branching bilaterian group(s). Recent
phylogenomic data suggested that Xenoturbellida and Acoelomorpha are
sister taxa and together constitute an early branch of Bilateria7. Here
we assemble three independent data setsmitochondrial genes, a
phylogenomic data set of 38,330 amino-acid positions and new microRNA
(miRNA) complements and show that the position of Acoelomorpha is
strongly affected by a long-branch attraction (LBA) artefact. When we
minimize LBA we find consistent support for a position of both
acoelomorphs and Xenoturbella within the deuterostomes. Themost likely
phylogeny links Xenoturbella and Acoelomorpha in a clade we call
Xenacoelomorpha. The Xenacoelomorpha is the sister group of the
Ambulacraria (hemichordates and echinoderms).We show that analyses of
miRNA complements8 have been affected by character loss in the acoels
and that both groups possess one miRNA and the gene Rsb66 otherwise
specific to deuterostomes. In addition,Xenoturbella shares one miRNA
with the ambulacrarians, and two with the acoels. This phylogeny makes
sense of the shared characteristics of Xenoturbellida andAcoelomorpha,
such as ciliary ultrastructure and diffuse nervous system, and implies
the loss of various deuterostome characters in the Xenacoelomorpha
including coelomic cavities, through gut and gill slits.
Nature, 470, 255-258 10 February 2011 doi:10.1038/nature09676 2010 Proteomic analysis of sea urchin (Strongylocentrotus purpuratus) spicule matrix. Mann K, Wilt FH, Poustka AJ. Background The sea urchin embryo has been an important model organism in developmental biology for more than a century. This is due to its relatively simple construction, translucent appearance, and the possibility to follow the fate of individual cells as development to the pluteus larva proceeds. Because the larvae contain tiny calcitic skeletal elements, the spicules, they are also important model organisms for biomineralization research. Similar to other biominerals the spicule contains an organic matrix, which is thought to play an important role in its formation. However, only few spicule matrix proteins were identified previously. Results Using mass spectrometry-based methods we have identified 231 proteins in the matrix of the S. purpuratus spicule matrix. Approximately two thirds of the identified proteins are either known or predicted to be extracellular proteins or transmembrane proteins with large ectodomains. The ectodomains may have been solubilized by partial proteolysis and subsequently integrated into the growing spicule. The most abundant protein of the spicule matrix is SM50. SM50-related proteins, SM30-related proteins, MSP130 and related proteins, matrix metalloproteases and carbonic anhydrase are among the most abundant components. Conclusions The spicule matrix is a relatively complex mixture of proteins not only containing matrix-specific proteins with a function in matrix assembly or mineralization, but also: 1) proteins possibly important for the formation of the continuous membrane delineating the mineralization space; 2) proteins for secretory processes delivering proteinaceous or non-proteinaceous precursors; 3) or proteins reflecting signaling events at the cell/matrix interface. Comparison of the proteomes of different skeletal matrices allows prediction of proteins of general importance for mineralization in sea urchins, such as SM50, SM30-E, SM29 or MSP130. The comparisons also help point out putative tissue-specific proteins, such as tooth phosphodontin or specific spicule matrix metalloproteases of the MMP18/19 group. Furthermore, the direct sequence analysis of peptides by MS/MS validates many predicted genes and confirms the existence of the corresponding proteins. Proteome Sci. 2010 Jun 17;8(1):33. Data integration using scanners with SQL output--the bioscanners project at sourceforge Groth D, Hartmann S, Friemel M, Hill N, Müller S, Poustka AJ, Panopoulou G There is currently no standardized
approach for parsing output that the numerous bioinformatical tools
generate. Because the framework approach of the Bio-toolkits has some
shortcomings, we searched for alternative approaches. To this end, we
evaluated scanner generators for various programming languages with
respect to their potential of standalone, small and fast applications
that can easily delivered on any modern and many ancient operating
system. We developed sample applications that generate standard SQL
database code and thereby greatly simplify the parsing work of data
integration and data analysis. At the sourceforge project page the
source code and some binaries for a selection of our applications are
freely available at http://bioscanners.sourceforge.net.
J Integr Bioinform. 2010 Mar 25;7(3). doi: 10.2390/biecoll-jib-2010-122. Phosphoproteomes of Strongylocentrotus purpuratus shell and tooth matrix: identification of a major acidic sea urchin tooth phosphoprotein, phosphodontin Karlheinz Mann , Albert J Poustka and Matthias Mann Background Sea urchin is a major model organism for developmental biology and biomineralization research. However, identification of proteins involved in larval skeleton formation and mineralization processes in the embryo and adult, and the molecular characterization of such proteins, has just gained momentum with the sequencing of the Strongylocentrotus purpuratus genome and the introduction of high-throughput proteomics into the field. Results The present report contains the determination of test (shell) and tooth organic matrix phosphoproteomes. Altogether 34 phosphoproteins were identified in the biomineral organic matrices. Most phosphoproteins were specific for one compartment, only two were identified in both matrices. The sea urchin phosphoproteomes contained several obvious orthologs of mammalian proteins, such as a Src family tyrosine kinase, protein kinase C-delta 1, Dickkopf-1 and other signal transduction components, or nucleobindin. In most cases phosphorylation sites were conserved between sea urchin and mammalian proteins. However, the majority of phosphoproteins had no mammalian counterpart. The most interesting of the sea urchin-specific phosphoproteins, from the perspective of biomineralization research, was an abundant highly phosphorylated and very acidic tooth matrix protein composed of 35 very similar short sequence repeats, a predicted N-terminal secretion signal sequence, and an Asp-rich C-terminal motif, contained in [Glean3:18919]. Conclusions The 64 phosphorylation sites determined represent the most comprehensive list of experimentally identified sea urchin protein phosphorylation sites at present and are an important addition to the recently analyzed Strongylocentrotus purpuratus shell and tooth proteomes. The identified phosphoproteins included a major, highly phosphorylated protein, [Glean3:18919], for which we suggest the name phosphodontin. Although not sequence-related to such highly phosphorylated acidic mammalian dental phosphoproteins as phosphoryn or dentin matrix protein-1, phosphodontin may perform similar functions in the sea urchin tooth. More than half of the detected proteins were not previously identified at the protein level, thus confirming the existence of proteins only known as genomic sequences previously. Proteome Science 2010, 8:6 2009 Deeply conserved chordate non-coding sequences preserve genome synteny but do not drive gene duplicate retention Hufton AL, Mathia S, Braun H, Georgi U, Lehrach H, Vingron M, Poustka AJ, and Panopoulou G. Animal genomes possess highly conserved cis-regulatory sequences that are often found near genes that regulate transcription and development. Researchers have proposed that the strong conservation of these sequences may affect the evolution of the surrounding genome, both by repressing rearrangement, and possibly by promoting duplicate gene retention. Conflicting data, however, have made the validity of these propositions unclear. Here, we use a new computational method to identify phylogenetically conserved non-coding elements (PCNEs) in a manner that is not biased by rearrangement and duplication. This method is powerful enough to identify more than a thousand PCNEs that have been conserved between vertebrates and the basal chordate amphioxus. We test 42 of our PCNEs in transgenic zebrafish assays---including examples from vertebrates and amphioxus---and find that the majority are functional enhancers. We find that PCNEs are enriched around genes with ancient synteny conservation, and that this association is strongest for extragenic PCNEs, suggesting that cis-regulatory interdigitation plays a key role in repressing genome rearrangement. Next, we classify mouse and zebrafish genes according to association with PCNEs, synteny conservation, duplication history, and presence in bidirectional promoter pairs, and use this data to cluster gene functions into a series of distinct evolutionary patterns. These results demonstrate that subfunctionalization of conserved cis-regulation has not been the primary determinate of gene duplicate retention in vertebrates. Instead, the data support the Gene Balance Hypothesis, which proposes that duplicate retention has been driven by selection against dosage imbalances in genes with many protein connections. Genome Res. 2009 Aug 24. [Epub ahead of print] Monte Carlo analysis of an ODE Model of the Sea Urchin Endomesoderm Network Kühn C, Wierling C, Kühn A, Klipp E, Panopoulou G, Lehrach H, and Poustka AJ. BACKGROUND: Gene Regulatory Networks (GRNs) control the differentiation, specification and function of cells at the genomic level. The levels of interactions within large GRNs are of enormous depth and complexity. Details about many GRNs are emerging, but in most cases it is unknown to what extent they control a given process, i.e. the grade of completeness is uncertain. This uncertainty stems from limited experimental data, which is the main bottleneck for creating detailed dynamical models of cellular processes. Parameter estimation for each node is often infeasible for very large GRNs. We propose a method, based on random parameter estimations through Monte-Carlo simulations to measure completeness grades of GRNs. RESULTS: We developed a heuristic to assess the completeness of large GRNs, using ODE simulations under different conditions and randomly sampled parameter sets to detect parameter-invariant effects of perturbations. To test this heuristic, we constructed the first ODE model of the whole sea urchin endomesoderm GRN, one of the best studied large GRNs. We find that nearly 48% of the parameter-invariant effects correspond with experimental data, which is 65% of the expected optimal agreement obtained from a submodel for which kinetic parameters were estimated and used for simulations. Randomized versions of the model reproduce only 23.5% of the experimental data. CONCLUSION: The method described in this paper enables an evaluation of network topologies of GRNs without requiring any parameter values. The benefit of this method is exemplified in the first mathematical analysis of the complete Endomesoderm Network Model. The predictions we provide deliver candidate nodes in the network that are likely to be erroneous or miss unknown connections, which may need additional experiments to improve the network topology. This mathematical model can serve as a scaffold for detailed and more realistic models. We propose that our method can be used to assess a completeness grade of any GRN. This could be especially useful for GRNs involved in human diseases, where often the amount of connectivity is unknown and/or many genes/interactions are missing. BMC Systems Biology 2009 Aug 23;3:83  Chordin is required for neural but not axial development in sea urchin embryos Cynthia Bradham, Catherine Oikonomou, Alexander Kühn, Amanda B. Core, Joshua W. Modell, David R. McClay, and Albert J. Poustka The oral-aboral (OA) axis in
the sea urchin is specified by the TGFß family members
Nodal and BMP2/4. Nodal promotes oral specification, whereas BMP2/4, despite being expressed in the oral territory, is required for aboral specification. This study explores the role of Chordin (Chd) during sea urchin embryogenesis. Chd is a secreted BMP inhibitor that plays an important role in axial and neural specification and patterning in Drosophila and vertebrate embryos. In L. variegatus embryos, Chd and BMP2/4 are functionally antagonistic. Both are expressed in overlapping domains in the oral territory prior to and during gastrulation. Perturbation shows that, surprisingly, Chd is not involved in OA axis specification. Instead, Chd is required both for normal patterning of the ciliary band at the OA boundary and for development of synaptotagmin B-positive (synB) neurons in a manner that is reciprocal with BMP2/4. Chd expression and synB-positive neural development are both downstream from p38 MAPK and Nodal, but not Goosecoid. These data are summarized in a model for synB neural development. Developmental Biology 2009, Volume 328, Issue 2, Pages 221-233
2008
In-depth, high-accuracy proteomics of sea urchin tooth matrix Mann K, Poustka AJ, Mann MBackground Results Conclusion
Ca(2+) signaling occurs via second messenger release from intraorganelle synthesis sites. Davis LC, Morgan AJ, Ruas M, Wong JL, Graeff RM, Poustka AJ,
Lee HC, Wessel GM, Parrington J, Galione A. Cyclic ADP-ribose is an important Ca(2+)-mobilizing cytosolic
messenger synthesized from beta-NAD(+) by ADP-ribosyl cyclases (ARCs).
However, the focus upon ectocellular mammalian ARCs (CD38 and CD157)
has led to confusion as to how extracellular enzymes generate
intracellular messengers in response to stimuli. We have cloned and
characterized three ARCs in the sea urchin egg and found that
endogenous ARCbeta and ARCgamma are intracellular and located within
the lumen of acidic, exocytotic vesicles, where they are optimally
active. Intraorganelle ARCs are shielded from cytosolic substrate and
targets by the organelle membrane, but this barrier is circumvented by
nucleotide transport. We show that a beta-NAD(+) transporter provides
ARC substrate that is converted luminally to cADPR, which, in turn, is
shuttled out to the cytosol via a separate cADPR transporter. Moreover,
nucleotide transport is integral to ARC activity physiologically
because three transport inhibitors all inhibited the
fertilization-induced Ca(2+) wave that is dependent upon cADPR. This
represents a novel signaling mechanism whereby an extracellular
stimulus increases the concentration of a second messenger by promoting
messenger transport from intraorganelle synthesis sites to the cytosol. Current
Biology 2008 Oct 28;18(20):1612-8.
Detlef Groth, Stefanie Hartmann, Georgia Panopoulou, Albert J.
Poustka and Steffen Hennig
Mann K, Poustka AJ, Mann M ABSTRACT: BACKGROUND: The organic
matrix
of biominerals plays an important role in biomineral formation and in
determining
biomineral properties. However, most components of biomineral matrices
remain unknown at present. In sea urchin, which is an important model
organism
for developmental biology and biomineralization, only few matrix
components
have been identified and characterized at the protein level. The recent
publication of the Strongylocentrotus purpuratus genome sequence
rendered
possible not only the identification of possible matrix proteins at the
gene level, but also the direct identification of proteins contained in
matrices of skeletal elements by in-depth, high-accuracy, proteomic
analysis.
RESULTS: We identified 110 proteins as components of sea urchin test
and
spine organic matrix. Fourty of these proteins occurred in both
compartments
while others were unique to their respective compartment. More than 95%
of the proteins were detected in sea urchin skeletal matrices for the
first
time. The most abundant protein in both matrices was the previously
characterized
spicule matrix protein SM50, but at least eight other members of this
group,
many of them only known as conceptual translation products previously,
were identified by mass spectrometric sequence analysis of peptides
derived
from in vitro matrix degradation. The matrices also contained proteins
implicated in biomineralization processes previously by inhibition
studies
using antibodies or specific enzyme inhibitors, such as matrix
metalloproteases
and members of the mesenchyme-specific MSP130 family. Other components
were carbonic anhydrase, collagens, echinonectin, a
alpha2-macroglobulin-like
protein and several proteins containing scavenger receptor
cysteine-rich
domains. A few possible signal transduction pathway components, such as
GTP-binding proteins, a semaphorin and a possible tyrosine kinase were
also identified. CONCLUSION: This report presents the most
comprehensive
list of sea urchin skeletal matrix proteins available at present. The
complex
mixture of proteins identified in matrices of the sea urchin skeleton
may
reflect many different aspects of the mineralization process. Because
LC-MS/MS-based
methods directly measures peptides our results validate many predicted
genes and confirm the existence of the corresponding proteins.
Considering
the many newly identified matrix proteins, this proteomic study may
serve
as a road map for the further exploration of biomineralization
processes
in an important model organism. Proteome
Science 2008, 6:22 (11 August 2008) Early vertebrate whole genome
duplications
were predated by a period of intense genome rearrangement Andrew L Hufton, Detlef Groth,
Martin Vingron,
Hans Lehrach, Albert J Poustka, and Georgia Panopoulou Researchers, supported by data from polyploid plants, have suggested that whole genome duplication (WGD) may induce genomic instability and rearrangement, an idea which could have important implications for vertebrate evolution. Benefiting from the newly released amphioxus genome sequence (Branchiostoma floridae), an invertebrate which researchers have hoped is representative of the ancestral chordate genome, we have used gene proximity conservation to estimate rates of genome rearrangement throughout vertebrates and some of their invertebrate ancestors. We find that, while amphioxus remains the best single source of invertebrate information about the early chordate genome, its genome structure is not particularly well conserved and it cannot be considered a fossilization of the vertebrate pre-duplication genome. In agreement with previous reports, we identify two WGD events in early vertebrates and another in teleost fish. However, we find that the early vertebrate WGD events were not followed by increased rates of genome rearrangement. Indeed, we measure massive genome rearrangement prior to these WGD events. We propose that the vertebrate WGD events may have been symptoms of a pre-existing predisposition toward genomic structural change. Evolution of a Core Gene Network for Skeletogenesis in Chordates Jochen Hecht, Sigmar Stricker,
Ulrike Wiecha,
Asita Stiege, Georgia Panopoulou, Lars Podsiadlowski, Albert J.
Poustka,
Christoph Dieterich, Siegfried Ehrich, Julia Suvorova, Stefan Mundlos,
Volkhard Seitz The skeleton is one of the most
important
features for the reconstruction of vertebrate phylogeny but few data
are
available to understand its molecular origin. In mammals the Runt genes
are central regulators of skeletogenesis. Runx2 was shown to be
essential
for osteoblast differentiation, tooth development, and bone formation.
Both Runx2 and Runx3 are essential for chondrocyte maturation.
Furthermore,
Runx2 directly regulates Indian hedgehog expression, a master
coordinator
of skeletal development. To clarify the correlation of Runt gene
evolution
and the emergence of cartilage and bone in vertebrates, we cloned the
Runt
genes from hagfish as representative of jawless fish (MgRunxA, MgRunxB)
and from dogfish as representative of jawed cartilaginous fish
(ScRunx1-3).
According to our phylogenetic reconstruction the stem species of
chordates
harboured a single Runt gene and thereafter Runt locus duplications
occurred
during early vertebrate evolution. All newly isolated Runt genes were
expressed
in cartilage according to quantitative PCR. In situ hybridisation
confirmed
high MgRunxA expression in hard cartilage of hagfish. In dogfish
ScRunx2
and ScRunx3 were expressed in embryonal cartilage whereas all three
Runt
genes were detected in teeth and placoid scales. In cephalochordates
(lancelets)
Runt, Hedgehog and SoxE were strongly expressed in the gill bars and
expression
of Runt and Hedgehog was found in endo- as well as ectodermal cells.
Furthermore
we demonstrate that the lancelet Runt protein binds to Runt binding
sites
in the lancelet Hedgehog promoter and regulates its activity. Together,
these results suggest that Runt and Hedgehog were part of a core gene
network
for cartilage formation, which was already active in the gill bars of
the
common ancestor of cephalochordates and vertebrates and diversified
after
Runt duplications had occurred during vertebrate evolution. The
similarities
in expression patterns of Runt genes support the view that teeth and
placoid
scales evolved from a homologous developmental module. PLoS
Genet 4(3) March 6 2008: e1000025. doi:10.1371/journal.pgen.1000025
2007 Modeling development: The
spikes of
the sea urchin Kühn C, Kühn A, Poustka AJ, Klipp E Modeling of specification events
during
development poses new challenges to biochemical modeling. These include
data limitations and a notorious absence of homeostasis in developing
systems.
The sea urchin is one of the best studied model organisms concerning
development
and a network, the Endomesoderm Network, has been proposed that is
presumed
to control endoderm and mesoderm specification in the embryo of
Strongylocentrotus
purpuratus. We have constructed a ynamic model of a subnetwork of the
Endomesoderm
Network. In constructing the model, we had to resolve the following
issues:
choice of appropriate subsystem, assignment of embryonic data to
cellular
model, choice of appropriate kinetics. Although the resulting model is
capable of reproducing fractions of the experimental data, it falls
short
of reproducing specification of cell types. These findings can
facilitate
the refinement of the Endomesoderm Network. Genome
Informatics 2007, 18(1), 75-84 A global view of gene
expression in
lithium and zinc treated sea urchin embryos: new components of gene
regulatory
networks Poustka AJ, Kühn A, Groth D, Weise V, Yaguchi S, Burke RD, Herwig R, Lehrach H, Panopoulou G. BACKGROUND: The genome of the sea
urchin
Strongylocentrotus purpuratus has recently been sequenced because it is
a major model system for the study of gene regulatory networks.
Embryonic
expression patterns for most genes are unknown, however. RESULTS: Using
large-scale screens on arrays carrying 50% to 70% of all genes, we
identified
novel territory-specific markers. Our strategy was based on
computational
selection of genes that are differentially expressed in lithium-treated
embryos, which form excess endomesoderm, and in zinc-treated embryos,
in
which endomesoderm specification is blocked. Whole-mount in situ
hybridization
(WISH) analysis of 700 genes indicates that the apical organ region is
eliminated in lithium-treated embryos. Conversely, apical and
specifically
neural markers are expressed more broadly in zinc-treated embryos,
whereas
endomesoderm signaling is severely reduced. Strikingly, the number of
serotonergic
neurons is amplified by at least tenfold in zinc-treated embryos. WISH
analysis further indicates that there is crosstalk between the Wnt
(wingless
int), Notch, and fibroblast growth factor signaling pathways in
secondary
mesoderm cell specification and differentiation, similar to signaling
cascades
that function during development of presomitic mesoderm in mouse
embryogenesis.
We provide differential expression data for more than 4,000 genes and
WISH
patterns of more than 250 genes, and more than 2,400 annotated WISH
images.
CONCLUSION: Our work provides tissue-specific expression patterns for a
large fraction of the sea urchin genes that have not yet been included
in existing regulatory networks and await functional integration.
Furthermore,
we noted neuron-inducing activity of zinc on embryonic development;
this
is the first observation of such activity in any organism Genome Biology
2007,
8:R85 doi:10.1186/gb-2007-8-5-r85 2006
The genome of the sea urchin Strongylocentrotus purpuratus Sea Urchin Genome Sequencing Consortium et. al. We report the sequence and
analysis of
the 814-megabase genome of the sea urchin Strongylocentrotus
purpuratus,
a model for developmental and systems biology. The sequencing strategy
combined whole-genome shotgun and bacterial artificial chromosome (BAC)
sequences. This use of BAC clones, aided by a pooling strategy,
overcame
difficulties associated with high heterozygosity of the genome. The
genome
encodes about 23,300 genes, including many previously thought to be
vertebrate
innovations or known only outside the deuterostomes. This echinoderm
genome
provides an evolutionary outgroup for the chordates and yields insights
into the evolution of deuterostomes. Science.
2006 Nov 10;314(5801):941-52 RTK and TGF-beta signaling pathways genes in the sea urchin genome Lapraz F, Rottinger E, Duboc V, Range R, Duloquin L, Walton K, Wu SY, Bradham C, Loza MA, Hibino T, Wilson K, Poustka A, McClay D, Angerer L, Gache C, Lepage T. The Receptor Tyrosine kinase
(RTK) and
TGF-beta signaling pathways play essential roles during development in
many organisms and regulate a plethora of cellular responses. From the
genome sequence of Strongylocentrotus purpuratus, we have made an
inventory
of the genes encoding receptor tyrosine kinases and their ligands, and
of the genes encoding cytokines of the TGF-beta superfamily and their
downstream
components. The sea urchin genome contains at least 20 genes coding for
canonical receptor tyrosine kinases. Seventeen of the nineteen
vertebrate
RTK families are represented in the sea urchin. Fourteen of these RTK
among
which ALK, CCK4/PTK7, DDR, EGFR, EPH, LMR, MET/RON, MUSK, RET, ROR,
ROS,
RYK, TIE and TRK are present as single copy genes while pairs of
related
genes are present for VEGFR, FGFR and INSR. Similarly, nearly all the
subfamilies
of TGF-beta ligands identified in vertebrates are present in the sea
urchin
genome including the BMP, ADMP, GDF, Activin, Myostatin, Nodal and
Lefty,
as well as the TGF-beta sensu stricto that had not been characterized
in
invertebrates so far. Expression analysis indicates that the early
expression
of nodal, BMP2/4 and lefty is restricted to the oral ectoderm
reflecting
their role in providing positional information along the oral-aboral
axis
of the embryo. The coincidence between the emergence of
TGF-beta-related
factors such as Nodal and Lefty and the emergence of the deuterostome
lineage
strongly suggests that the ancestral function of Nodal could have been
related to the secondary opening of the mouth which characterizes this
clade, a hypothesis supported by functional data in the extant species.
The sea urchin genome contains 6 genes encoding TGF-beta receptors and
4 genes encoding prototypical Smad proteins. Furthermore, most of the
transcriptional
activators and repressors shown to interact with Smads in vertebrates
have
orthologues in echinoderms. Finally, the sea urchin genome contains an
almost complete repertoire of genes encoding extracellular modulators
of
BMP signaling including Chordin, Noggin, Sclerotin, SFRP, Gremlin, DAN
and Twisted gastrulation. Taken together, these findings indicate that
the sea urchin complement of genes of the RTK and TGF-beta signaling
pathways
is qualitatively very similar to the repertoire present in vertebrates,
and that these genes are part of the common genetool kit for
intercellular
signaling of deuterostomes. Dev
Biol. 2006 Dec 1;300(1):132-52. Epub 2006 Aug 24. Gene identification and analysis of transcripts differentially regulated in fracture healing by EST sequencing in the domestic sheep. Hecht J, Kuhl H, Haas SA, Bauer S, Poustka AJ, Lienau J, Schell H, Stiege AC, Seitz V, Reinhardt R, Duda GN, Mundlos S, Robinson PN. BACKGROUND: The sheep is an important model animal for testing novel fracture treatments and other medical applications. Despite these medical uses and the well known economic and cultural importance of the sheep, relatively little research has been performed into sheep genetics, and DNA sequences are available for only a small number of sheep genes. RESULTS: In this work we have sequenced over 47 thousand expressed sequence tags (ESTs) from libraries developed from healing bone in a sheep model of fracture healing. These ESTs were clustered with the previously available 10 thousand sheep ESTs to a total of 19087 contigs with an average length of 603 nucleotides. We used the newly identified sequences to develop RT-PCR assays for 78 sheep genes and measured differential expression during the course of fracture healing between days 7 and 42 postfracture. All genes showed significant shifts at one or more time points. 23 of the genes were differentially expressed between postfracture days 7 and 10, which could reflect an important role for these genes for the initiation of osteogenesis. CONCLUSION: The sequences we have identified in this work are a valuable resource for future studies on musculoskeletal healing and regeneration using sheep and represent an important head-start for genomic sequencing projects for Ovis aries, with partial or complete sequences being made available for over 5,800 previously unsequenced sheep genes. 2005
Timing and mechanism of ancient vertebrate genome duplications - the adventure of a hypothesis Panopoulou G and Poustka AJ Complete genome doubling has
long-term
consequences for the genome structure and the subsequent evolution of
an
organism. It has been suggested that two genome duplications occurred
at
the origin of vertebrates (known as the 2R hypothesis). However, there
has been considerable debate as to whether these were two successive
duplications,
or whether a single duplication occurred, followed by large-scale
segmental
duplications. In this article, we review and compare the evidence for
the
2R duplications from vertebrate genomes with similar data from other
more
recent polyploids. Trends
Genet. 2005 Aug 11; [Epub ahead of print] 2004 Nodal/activin signaling establishes oral-aboral polarity in the early sea urchin embryo Vera Lynn Flowers , Girard R. Courteau , Albert J. Poustka , Wei Weng , Judith M. Venuti Components of the Wnt signaling pathway are involved in patterning the sea urchin primary or animal-vegetal (AV) axis, but the molecular cues that pattern the secondary embryonic axis, the aboral/oral (AO) axis, are not known. In an analysis of signaling molecules that influence patterning along the sea urchin embryonic axes, we found that members of the activin subfamily of transforming growth factor- (TGF-) signaling molecules influence the establishment of AO polarities in the early embryo. Injection of activin mRNAs into fertilized eggs or treatment with exogenously applied recombinant activin altered the allocation of ectodermal fates and ventralized the embryo. The phenotypes observed resemble the ventralized phenotype previously reported for NiCl2, a known disrupter of AO patterning. Sensitivity to exogenous activin occurs between fertilization and the late blastula stage, which is also the time of highest NiCl2 sensitivity. These results argue that specification of fates along the embryonic AO axis involves TGF- signaling. To further examine TGF- signaling in these embryos, we cloned an endogenous TGF- from sea urchin embryos that is a member of the activin subfamily, SpNodal, and show through gain of function analysis that it recapitulates results obtained with exogenous activins and NiCl2. The expression pattern of SpNodal is consistent with a role for nodal signaling in the establishment of fates along the AO axis. Loss of function experiments using SpNodal antisense morpholinos also support a role for SpNodal in the establishment of the AO axis. Developmental
Dynamics 231: 727-740, 2004 On the origin of the chordate central nervous system: expression of onecut in the sea urchin embryo Albert J. Poustka, Alexander Kühn, Vesna Radosavljevic, Ruth Wellenreuther, Hans Lehrach, and Georgia Panopoulou We have identified a transcription factor of the onecut class in the sea urchin Strongylocentrotus purpuratus that represents an orthologue of the mammalian gene HNF6, the founding member of the onecut class of proteins. The isolated sea urchin gene, named SpOnecut, encodes a protein of 483 amino acids with one cut domain and a homeodomain. Phylogenetic analysis clearly places the sea urchin gene into this family, most closely related to the ascidian onecut gene HNF-6. Nevertheless phylogenetic analysis reveals a difficult phylogeny indicating that certain members of the family evolve more rapidly than others and also that the cut domain and homeodomain evolve at a different pace. In fly, worm, ascidian and teleost fish the onecut genes isolated so far are exclusively expressed in cells of the central nervous system, whereas in mammals the 2 copies of the gene have acquired additional functions in liver and pancreas development. In the sea urchin embryo, expression is first detected in the emerging ciliary band at the late blastula stage. During the gastrula stage, expression is limited to the ciliary band. In the early pluteus stage SpOnecut is expressed at the apical organ and the elongating arms but continues most prominently in the ciliary band. This is the first gene known that exclusively marks the ciliary band and therein the apical organ in a pluteus larva, whereas chordate orthologs execute essential functions in dorsal central nervous system (CNS) development. The significance of this finding for the hypothesis that the ciliary bands and apical organs of the hypothetical "dipleurula" like chordate ancestor and the chordate/vertebrate CNS are of common origin is discussed Evolution
and Development 6:4, 227-236 Expression of an NK2 homeodomain gene in the apical ectoderm defines a new territory in the early sea urchin embryo. Takacs CM, Amore G, Oliveri P, Poustka AJ, Wang D, Burke RD, Peterson KJ. We have identified an NK2 family homeodomain transcription factor, SpNK2.1, in the sea urchin Strongylocentrotus purpuratus whose transcripts are initially detected within the apical plate ectoderm of the hatching blastula and are confined to the apical organ at least through 2 weeks of development. Protein localization studies demonstrate that SpNK2.1 is restricted to the apical plate epithelium, but is excluded from the nucleus of serotonergic neurons. The expression profile of SpNK2.1 is dictated via two separate regulatory systems. Initially, SpNK2.1 is restricted to the apical pole domain by beta-catenin-dependent processes operating along the animal-vegetal axis, as evidenced by an expansion of SpNK2.1 expression upon cadherin overexpression. Starting at gastrulation, expression in the apical plate is maintained by SpDri, the sea urchin orthologue of dead ringer. Abrogation of SpDri results in the downregulation of SpNK2.1 after gastrulation, but SpDri is not necessary for the initial activation of SpNK2.1. Loss of function experiments using SpNK2.1-specific morpholino antisense oligonucleotides and SpNK2.1 overexpression experiments do not disrupt embryonic development and have no effect upon the development of neuronal components of the apical organ. Nonetheless, SpNK2.1 defines a new early territory of the sea urchin embryo. Dev Biol. 2004 May
1;269(1):152-64. Cloning of a novel phospholipase c-d isoform from pacific purple sea urchin (Strongylocentrotus purpuratus) gametes and its expression during early embryonic development Kevin Coward*, Helen Owen*, Albert J. Poustka, Olivia Hibbitt, Richard Tunwell, Hiroki Kubota, Karl Swann and John Parrington * Both Authors contributed equally to this work Calcium (Ca2+) is a ubiquitous
intracellular
messenger, controlling a diverse range of cellular processes, including
fertilization and development of the embryo. One of the key mechanisms
involved in triggering intracellular calcium release is the generation
of the second messenger inositol-1,4,5-phosphate (IP3) by the
phospholipase
C (PLC) class of enzymes. Although five distinct forms of PLC have been
identified in mammals (ß, d, g, e and z), only one, PLCg, has
thus
far been detected in echinoderms. In the present study, we describe the
isolation of a cDNA encoding a novel PLC isoform of the delta (d)
subclass,
PLC-dsu, from the sperm and egg cytosol of the Pacific purple sea
urchin
Strongylocentrotus purpuratus. The PLC-dsu cDNA (2.44kb) encodes a 742
amino acid polypeptide with an open reading frame of 84.6kDa and a pI
of
6.04. All of the characteristic domains found in mammalian PLCd
isoforms
(PH domain, EF hands, an X-Y catalytic region, and a C2 domain) are
present
in PLC-dsu. A homology search revealed that PLC-dsu shares most
sequence
identity with bovine PLC-d2 (39%). We present evidence that PLC-dsu is
expressed in unfertilized eggs, fertilized eggs, and during the early
embryo.
In addition to Northern and Polymerase Chain Reaction (PCR) analysis,
In
situ hybridization experiments further demonstrated that the embryonic
regions within which the PLC-dsu transcript can be detected during
early
embryonic development, are associated with the highest levels of
proliferative
activity, suggesting a possible involvement with metabolism or cell
cycle
regulation. Biochemical and Biophysical
Research
Communications 313 (2004): 891-898 2003 Generation, annotation, evolutionary analysis and database integration of 20,000 unique sea urchin EST clusters Albert J. Poustka, Detlef Groth, Steffen Hennig, Sabine Thamm, Andrew Cameron, Alfred Beck, Richard Reinhardt, Ralf Herwig, Georgia Panopoulou and Hans Lehrach Together with the hemichordates, sea urchins represent basal groups of nonchordate invertebrate deuterostomes that occupy a key position in bilaterian evolution. Because sea urchin embryos are also amenable to functional studies the sea urchin system has emerged as one of the leading models for the analysis of the function of genomic regulatory networks that control development. We have analysed a total of 107,283 cDNA clones of libraries that span the development of the sea urchin Strongylocentrotus purpuratus. Normalisation by oligonucleotide fingerprinting, EST sequencing and sequence clustering resulted in an EST catalogue comprised of 20,000 unique genes or gene fragments. Around 7000 of the unique EST consensus sequences were associated with molecular and developmental functions. Phylogenetic comparison of the identified genes to the genome of the urochordate Ciona intestinalis indicate that at least one quarter of the genes thought to be chordate specific were already present at the basis of deuterostome evolution. Comparison of the number of gene copies in sea urchin to those in chordates and vertebrates indicates that the sea urchin genome has not undergone extensive gene or complete genome duplications. The established unique gene set represents an essential tool for the annotation and assembly of the forthcoming sea urchin genome sequence. Genome Research 13:2736-2746, 2003 New evidence for genome-wide duplications at the origin of vertebrates using an amphioxus gene set and completed animal genomes Panopoulou G, Hennig S, Groth D, Krause A, Poustka AJ, Herwig R, Vingron M, Lehrach H. The 2R hypothesis predicting two genome duplications at the origin of vertebrates is highly controversial. Studies published so far include limited sequence data from organisms close to the hypothesized genome duplications. Through the comparison of a gene catalog from amphioxus, the closest living invertebrate relative of vertebrates, to 3453 single-copy genes orthologous between Caenorhabditis elegans (C), Drosophila melanogaster (D), and Saccharomyces cerevisiae (Y), and to Ciona intestinalis ESTs, mouse, and human genes, we show with a large number of genes that the gene duplication activity is significantly higher after the separation of amphioxus and the vertebrate lineages, which we estimate at 650 million years (Myr). The majority of human orthologs of 195 CDY groups that could be dated by the molecular clock appear to be duplicated between 300 and 680 Myr with a mean at 488 million years ago (Mya). We detected 485 duplicated chromosomal segments in the human genome containing CDY orthologs, 331 of which are found duplicated in the mouse genome and within regions syntenic between human and mouse, indicating that these were generated earlier than the human-mouse split. Model based calculations of the codon substitution rate of the human genes included in these segments agree with the molecular clock duplication time-scale prediction. Our results favor at least one large duplication event at the origin of vertebrates, followed by smaller scale duplication closer to the bird-mammalian split. Genome
Research
13:1056-1066, 2003 A single amphioxus and sea urchin runt-gene suggests that runt-gene duplications occurred in early chordate evolution Stricker S*, Poustka AJ*, Wiecha U, Stiege A, Hecht J, Panopoulou G, Vilcinskas A, Mundlos S, Seitz V. * Both Authors contributed equally to this work Runt-homologous molecules are
characterized
by their DNA binding runt-domain which is highly conserved within
bilaterians.
The three mammalian runt-genes are master regulators in cartilage/bone
formation and hematopoiesis. Historically these features evolved in
Craniota
and might have been promoted by runt-gene duplication events. The
purpose
of this study was therefore to investigate how many runt-genes exist in
the stem species of chordates, by analyzing the number of runt-genes in
what is likely to be the closest living relative of Craniota-amphioxus.
To acquire further insight into the possible role of runt-genes in
early
chordate evolution we have determined the number of runt-genes in sea
urchins
and have analyzed the runt-expression pattern in this species.Our
findings
demonstrate the presence of a single runt-gene in amphioxus and sea
urchin,
which makes it highly likely that the stem species of chordates
harbored
only a single runt-gene. This suggests that runt-gene duplications
occurred
later in chordate phylogeny, and are possibly also associated with the
evolution of features such as hematopoiesis, cartilage and bone
development.In
sea urchin embryos runt-expression involves cells of endodermal,
mesodermal
and ectodermal origin. This complex pattern of expression might reflect
the multiple roles played by runt-genes in mammals. A strong
runt-signal
in the gastrointestinal tract of the sea urchin is in line with
runt-expression
in the intestine of nematodes and in the murine gastrointestinal tract,
and seems to be one of the phylogenetically ancient runt-expression
domains. Developmental
and Comparative Immunology, Volume 27, Issue 8, September 2003, Pages
673-684 2002 brachyury Target genes in the early sea urchin embryo isolated by differential macroarray screening Rast JP, Cameron RA, Poustka AJ, Davidson EH Brachyury is a transcription factor that functions in gastrulation and endoderm development throughout the Bilateria. Here, we identify genes that are expressed downstream of brachyury during gastrulation of the sea urchin embryo. Screens with two different complex probes generated by subtractive hybridization were carried out on high-density arrays of embryonic cDNA libraries. An mRNA sequence population from embryos expressing brachyury at its peak stage of expression was subtracted with message sequence from embryos in which Brachyury function had been "knocked-out" by injection of a morpholine-substituted antisense oligonucleotide to generate a differential probe for brachyury target genes. Another probe was made by using an mRNA population from embryos that mis-express brachyury at a stage just prior to the normal onset of expression, subtracted with message sequence taken from normal embryos at this stage. Screens carried out with these probes target overlapping but distinct sets of downstream genes. After partial sequence characterization, promising genes were independently analyzed by quantitative real-time PCR and by in situ hybridization. Two major classes of genes emerge in this study: genes expressed in the subset of the secondary mesenchyme cells (SMC) that will become pigment cells, and genes that are expressed in portions of the endoderm coincident with brachyury expression. The latter genes are candidates for direct transcriptional targets of Brachyury. Some of the endodermal genes that respond to Brachyury are cytoskeletal modulators that may play a role in gut morphogenesis. This finding is nsistent with the block in gastrulation induced by interfering with Brachyury function in sea urchins, and with known or suggested brachyury function in other species. Other endodermal target genes are expressed in the archenteron and might be terminal differentiation enzymes of the gut. Brachyury expression patterns for Strongylocentrotus purpuratus reported in this paper are entirely consistent with data from other echinoderm species. Brachyury expression in the vegetal plate is confined to the presumptive endodermal cells. Therefore, the SMC genes are likely to be indirect targets of Brachyury-induced signaling from the surrounding endoderm to the central mesoderm, or the effects on these genes may be indirect consequences of gross disruption of the vegetal plate. These results and other data suggest that the brachyury gene transduces information about the state of endodermal specification to genes that modulate morphogenesis and genes that perform terminal functions in the gut. Developmental
Biology, Volume 246, Issue 1, 1 June 2002, Pages 191-208 2000 A sea urchin genome project: sequence scan, virtual map, and additional resources Cameron RA, Mahairas G, Rast JP, Martinez P, Biondi TR, Swartzell S, Wallace JC, Poustka AJ, Livingston BT, Wray GA, Ettensohn CA, Lehrach H, Britten RJ, Davidson EH, Hood L Results of a first-stage Sea Urchin Genome Project are summarized here. The species chosen was Strongylocentrotus purpuratus, a research model of major importance in developmental and molecular biology. A virtual map of the genome was constructed by sequencing the ends of 76,020 bacterial artificial chromosome (BAC) recombinants (average length, 125 kb). The BAC-end sequence tag connectors (STCs) occur an average of 10 kb apart, and, together with restriction digest patterns recorded for the same BAC clones, they provide immediate access to contigs of several hundred kilobases surrounding any gene of interest. The STCs survey >5% of the genome and provide the estimate that this genome contains approximately 27,350 protein-coding genes. The frequency distribution and canonical sequences of all middle and highly repetitive sequence families in the genome were obtained from the STCs as well. The 500-kb Hox gene complex of this species is being sequenced in its entirety. In addition, arrayed cDNA libraries of >10(5) clones each were constructed from every major stage of embryogenesis, several individual cell types, and adult tissues and are available to the community. The accumulated STC data and an expanding expressed sequence tag database (at present including >12, 000 sequences) have been reported to GenBank and are accessible on public web sites. Proc.
Natl. Acad. Sci. USA, Vol. 97, Issue 17, 9514-9518, August 15, 2000
1999 Toward the gene catalogue of sea urchin development: the construction and analysis of an unfertilized egg cDNA library highly normalized by oligonucleotide fingerprinting. Poustka AJ, Herwig R, Krause A, Hennig S, Meier-Ewert S, Lehrach H We describe the use of
oligonucleotide
fingerprinting for the generation of a normalized cDNA library from
unfertilized
sea urchin eggs and report the preliminary analysis of this library,
which
resulted in the establishment of a partial gene catalogue of the sea
urchin
egg. In an analysis of 21,925 cDNA clones by hybridization with 217
oligonucleotide
probes, we were able to identify 6291 clusters corresponding to
different
transcripts, ranging in size from 1 to 265 clones. This corresponds to
an average 3.5-fold normalization of the starting library. The
normalized
library represents about one-third of all genes expressed in the sea
urchin
egg. To generate sequence information for the transcripts represented
by
the clusters, representative clones selected from 711 clusters
were
sequenced. The construction and preliminary analysis of the normalized
library are the first steps in the assembly of an increasingly complete
collection of maternal genes expressed in the sea urchin egg, which
will
provide a number of insights into the early development of this
well-characterized
model organism. Genomics
1999 Jul 15;59(2):122-33 Large-scale clustering of cDNA-fingerprinting data. Herwig R, Poustka AJ, Muller C, Bull C, Lehrach H, O'Brien J Max-Planck Institut fur Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany. herwig@molgen.mpg.de Clustering is one of the main mathematical challenges in large-scale gene expression analysis. We describe a clustering procedure based on a sequential k-means algorithm with additional refinements that is able to handle high-throughput data in the order ofhundreds of thousands of data items measured on hundreds of variables. The practical motivation for our algorithm is oligonucleotide fingerprinting-a method for simultaneous determination of expression level for every active gene of a specific tissue-although the algorithm can be applied as well to other large-scale projects like EST clustering and qualitative clustering of DNA-chip data. As a pairwise similarity measure between two p-dimensional data points, x and y, we introduce mutual information that can be interpreted as the amount of information about x in y, and vice versa. We show that for our purposes this measure is superior to commonly used metric distances, for example, Euclidean distance. We also introduce a modified version of mutual information as a novel method for validating clustering results when the true clustering is known. The performance of our algorithm with respect to experimental noise is shown by extensive simulation studies. The algorithm is tested on a subset of 2029 cDNA clones coming from 15 different genes from a cDNA library derived from human dendritic cells. Furthermore, the clustering of these 2029 cDNA clones is demonstrated when the entire set of 76,032 cDNA clones is processed. Genome
Research
1999 Nov;9(11):1093-105
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