Carnegie Institution of Washington, Department of Embryology, Baltimore,
MD 21210, USA
Present address: University of Utah School of Medicine, Department of
Neurobiology and Anatomy, Salt Lake City, UT 84132-3401, USA
Present address: University of Illinois at Urbana-Champaign, Department of
Cell and Structural Biology, Urbana-Champaign, IL 61801, USA
Author for correspondence (e-mail:
sanchez{at}neuro.utah.edu)
Accepted 11 August 2002
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SUMMARY |
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Key words: Regeneration, Stem cells, Plasticity, Database
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INTRODUCTION |
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Moreover, many members of this phylum possess large populations of
undifferentiated mesenchymal stem cells, the study of which could contribute
significantly to fundamental biomedical research in the areas of tissue
regeneration, stem cell maintenance and degenerative disorders. In most
free-living species these stem cells, which are often referred to as
neoblasts, are used for the regeneration of missing body parts and/or the
replacement of cells that are lost during the course of physiological turnover
(Gschwentner et al., 2001;
Ladurner et al., 2000
;
Newmark and Sánchez Alvarado,
2000
). Similarly, free mesenchymal cells in parasitic flukes are
known to produce complete larval forms
(Brusca and Brusca, 1990
;
Hyman, 1951
), and in the
cestode Taenia crassiceps complete cysts can be reconstituted from
individual cells (Toledo et al.,
1997
). Thus, platyhelminthes also provide a unique opportunity for
studying the mechanisms that underlie the control of cellular
pluripotentiality.
To address many of these unsolved problems, we and others
(Agata and Watanabe, 1999) have
chosen to reintroduce the freshwater planarian as an experimental model. We
report the establishment of a clonal line of a diploid, asexual form of the
planarian Schmidtea mediterranea (Turbellaria, Tricladida), along
with the isolation and sequence characterization of
3000 non-redundant,
expressed sequence tags (ESTs) from this organism. Furthermore, we show the
suitability of using planarians for high-throughput mapping of gene expression
patterns in the whole animal, and introduce the S.
mediterranea Database (SmedDb) in which the primary data,
computational analyses and expression data reside
(http://planaria.neuro.utah.edu).
RNA interference (Sánchez Alvarado
and Newmark, 1999
) and the ability to label the S.
mediterranea neoblasts specifically
(Newmark and Sánchez Alvarado,
2000
) will permit the identification and characterization of genes
involved in regenerative processes, ranging from the control of stem cell
proliferation and differentiation to the regulation of polarity, growth, scale
and proportion.
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MATERIALS AND METHODS |
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cDNA library preparation
Heads and 2-3 day regeneration blastemas were isolated from individuals of
asexual clonal line CIW4. Amputated tissue was immediately frozen in liquid
nitrogen and stored at -80°C until use. Total RNA was isolated using
TriZol reagent (BRL/Life Technologies); poly(A)+ RNA was prepared
using oligo d(T) cellulose (BRL/Life Technologies). Standard procedures were
used to synthesize and size-select the oligo d(T) primed cDNAs; the resulting
cDNAs were directionally cloned into the EcoRI and XhoI
restriction sites of pBluescript II SK (+) and electroporated into DH10B
cells. Unamplified cDNA libraries were replica plated on nitrocellulose
filters and grown on LB/carbenicillin plates. One set of replicate filters was
used for hybridization to identify abundant clones; these were excluded from
subsequent rounds of analysis. The second set of filters served as master
filters for recovery of non-redundant clones; these filters were stored on
LB/glycerol plates at -80°C. Non-redundant clones were picked and grown
overnight at 37°C in Magnificent Broth (MacConnell Research) with 100
µg/ml carbenicillin. Plasmid isolations were performed using a MiniPrep24
machine (MacConnell Research).
Sequence analysis, bioinformatics and the S. mediterranea
EST database (SmedDb)
Sequencing reactions were performed using Big Dye Terminator chemistry and
the resulting products were run on an ABI Prism 377 DNA sequencer. The
sequencing strategy is outlined in Fig.
1A. Obtained sequences were compared against one another using
stand alone BLAST (Altschul et al.,
1990) as a way to measure internal redundancy and to identify
unique clones. Statistical analysis of the frequency distribution of unique
sequences indicates that the non-redundant clones identified represent a
significant proportion of the complexity of the libraries (50-55%; see
http://planaria.neuro.utah.edu
for details). In order to allow the management and internet browser
accessibility of the data, unique sequences were deposited in a server
database running Cold Fusion 4.5 (Allaire) and batch analyzed at GenBank for
homology comparisons using BLASTc13 running either nucleotide-nucleotide
(BLASTn) or translated (BLASTx) searches. In addition, dbEST was also queried
using BLASTn, BLASTx and tBLASTx. Given the number of new sequences being
continuously deposited into GenBank, SmedDb has been programmed to update the
BLAST results for all planarian ESTs on command and/or automatically once a
week.
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Whole-mount in situ hybridization
Planarians (3-5 mm in length) and starved for at least 1 week were treated
with 2% HCl for 5 minutes on ice, and then fixed on ice for 2 hours in
Carnoy's fixative (EtOH:CHCl3:acetic acid, 6:3:1)
(Umesono et al., 1997). After
1 hour in methanol at -20°C, the planarians were bleached in 6%
H2O2 in methanol at room temperature. Bleached
planarians were loaded into incubation columns in an Insitu Pro hybridization
robot (Abimed/Intavis, Germany) and processed as described
(Sánchez Alvarado and Newmark,
1999
) with modifications to accommodate the liquid handling
characteristics of the machine.
GenBank accession numbers
GenBank accession numbers were: AY066058-AY066260; AY066262-AY066313;
AY066315-AY066438; AY066440-AY067204; AY067206-AY068336; AY068339-AY068349;
and AY068675-AY069025.
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RESULTS |
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The high percentage of planarian ESTs with putative orthologs in the public databases allowed us to further organize SmedDb into functional categories (Fig. 1B). The categories employed are derived from the expressed gene anatomy database (EGAD; http://www.tigr.org) and the gene ontology (http://www.geneontology.org) functional classification systems. Each entry in SmedDb consists of the cDNA name, similarity description and expression pattern, if available (see below). Selecting an entry in the database provides additional information such as the sequence sent for analysis, the assigned functional category, in situ hybridization data and the corresponding BLAST results linked to Entrez-PubMed (see http://planaria.neuro.utah.edu). Examples of SmedDb entries placed into functional categories are shown in Table 1. At least 77 transcription factors, 130 DNA replication/modification molecules and 97 receptors, channels and other membrane-associated proteins were putatively identified.
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Interestingly, when the planarian ESTs with significant homologies to
GenBank are ranked by lowest expectancy value, we find that 64% of the entries
in SmedDb have highest overall similarities to vertebrate rather than to
invertebrate sequences (Fig.
1C). When comparative BLASTx analyses between SmedDb and the
proteomes of C. elegans, D. melanogaster and H. sapiens were
performed, a set of 124 S. mediterranea ESTs with significant
similarity only to proteins found in the human genome were revealed.
Sixty-three of these are similar to human genes encoding proteins of unknown
function. Noteworthy is the presence in S. mediterranea of thymidine
phosphorylase/endothelial cell growth factor 1 (BLASTx
E=5x10-30), acyl-CoA dehydrogenase (BLASTx
E=2x10-21), epoxide hydrolase (BLASTx
E=5x10-29) and formiminotransferase cyclodeaminase (BLASTx
E=4x10-42). These genes were recently postulated to be
present in the human genome as a result of direct horizontal gene transfer
(HGT) between bacteria and vertebrates based on their absence in the genomes
of C. elegans and D. melanogaster
(Lander et al., 2001).
However, the presence of these transcripts in planarians suggests that these
loci are most probably not shared by bacteria and vertebrates via HGT, but
rather by descent through common ancestry
(Kyrpides and Olsen, 1999
;
Stanhope et al., 2001
).
High-throughput in situ hybridization
The 3000 independent ESTs available in SmedDb provide a wealth of
material for studying the flatworms. One such use will be for identifying cell
type- and region-specific markers. Thus, we have used whole-mount in situ
hybridization to begin to determine the spatial expression patterns of SmedDb
entries; to date, results from nearly 300 clones have been deposited in
SmedDb, and more are being added regularly as they become available. The
analysis has revealed some surprising complexities in the spatial expression
patterns of many of the genes represented in the EST collection
(Fig. 2). We find, for example,
that the morphologically simple cephalic ganglia of flatworms display a
diverse array of expression domains, some of which are depicted in
Fig. 2A (see figure legend for
explanation). In addition, other organ-system-specific genes have been
identified that label the gastrovascular system, the dorsal epithelium, the
excretory system and the pharynx (Fig.
2B from top to bottom). We also find transcripts expressed in
various subsets of cells, including the planarian neoblasts in which piwi, a
transcript found in many metazoan stem cells
(Benfey, 1999
), can be detected
(Fig. 2C, bottom picture).
Striking expression patterns defining both dorsal and ventral boundaries have
been observed as well. This is illustrated by the lateral view of in situ
hybridization results using clone H.8.1f, which has no known homolog in the
available databases (Fig.
2D).
|
Cell loss during de-growth
The identification of cell type-specific markers from the large-scale in
situ hybridization screen provides new tools for studying morphallaxis, a
classic problem first defined by Morgan in 1898
(Morgan, 1898). Morphallaxis
refers to the remodeling that occurs when small fragments of planarians (or
other organisms, like Hydra) restore their appropriate proportion and pattern
without adding additional tissue. In addition to this remodeling during
regenerative events, planarians show a high degree of plasticity in their
ability to either grow or de-grow, depending upon environmental conditions.
During periods of prolonged starvation, planarians will shrink
(Lillie, 1900
;
Schultz, 1904
;
Berninger, 1911
;
Child, 1911
;
Abeloos, 1930
): a 20 mm long
worm can be reduced to less than 1 mm over the course of several months. This
change in body size is due to an overall reduction in total cell number, as
opposed to a reduction in cell size
(Baguñà and Romero,
1981
; Romero and
Baguñà, 1991
). Previous studies of this phenomenon
have used techniques in which planarians are macerated into a suspension of
individual cells. Using this method, roughly 13 different cell types from
organisms in varying stages of growth and de-growth were classified and
quantitated (Baguñà and
Romero, 1981
; Romero and
Baguñà, 1991
). Because the flatworms were
dissociated into single cells in these studies, the distribution of the cells
could not be monitored in the whole animal as it changed in size. Furthermore,
the morphological criteria alone underestimated the true number of different
cell types in the planarian.
cDNA clone H112.3c shows weak sequence similarity to degenerin 1 from
C. elegans and is expressed in a subset of cells near the anterior
margin of the planarian (Fig.
3A); these cells are likely to be involved in chemoreception
through ciliated pits that lie at the ciliated anterior margin in this genus
(Farnesi and Tei, 1980). The
number of H112.3c-expressing cells can be counted easily in organisms of
different sizes after whole-mount in situ hybridization. Remarkably, the
number of these cells increases linearly with length
(Fig. 3B), suggesting that even
for cell types comprising a small percentage of the body (
0.03%), their
total numbers are regulated as the animal grows and shrinks. How these
organisms can `count' different cell types relative to total body size remains
a complete mystery.
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DISCUSSION |
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The establishment of a clonal line (CIW4) of the freshwater planarian
S. mediterranea and the identification of nearly 3000 non-redundant
cDNAs from this line will aid the molecular study of the most salient
biological properties of this taxon. Nearly 70% of all S.
mediterranea clones share significant homologies to sequences deposited
in GenBank (Fig. 1B), and a
large number of these have highest similarity to the deuterostome branch of
the metazoans (Fig. 1C). These
results indicate either a closer proximity of the phylum to the deuterostome
lineage as recently proposed by Tyler
(Tyler, 2001), or are more
likely a reflection of the poor representation of invertebrate sequences in
current databases. The latter possibility is illustrated by the identification
in planarians of cDNAs encoding proteins that until recently were ascribed to
be present only in bacteria and vertebrates based on a comparative analysis of
the human, fly and nematode genomes
(Lander et al., 2001
). The
presence of Thymidine phosphorylase/endothelial cell growth factor 1, acyl-CoA
dehydrogenase, epoxide hydrolase and formiminotransferase cyclodeaminase in
S. mediterranea suggests that these loci reached the vertebrates by
common ancestry and not by horizontal gene transfer as originally proposed
(Lander et al., 2001
).
Therefore, even though the proteomes of both C. elegans and D.
melanogaster have been deposited in GenBank, limiting sequence
comparisons to these two invertebrates is not sufficient to draw sound
phylogenetic conclusions, especially on the basis of BLAST results alone. Only
rigorous phylogenetic analyses can most closely approximate phyletic
relationships and we expect that the sequences in SmedDb will contribute to
the production of higher resolution intra- and inter-phyletic metazoan
relationships.
In addition to sharing a large number of genes with the human, fly and nematode genomes, it should be noted that several planarian cDNAs with significant similarities to human sequences were not identified in the C. elegans or D. melanogaster genomes by BLAST searches. At least 63 of these cDNAs are similar to human genes encoding proteins of unknown function. Therefore, S. mediterranea is likely to expand and complement the repertoire of organisms used for the study of genes and pathways involved in various aspects of human biology and disease.
The high-throughput in situ hybridization analyses reported here will serve
as a first step in deciphering the roles of genes encoding proteins of unknown
function. The tissue- or cell type-specific expression patterns of these genes
may provide hints as to their function. For example, cDNA clones H.14.5b and
H.12.6g share similarity with human genes for which no function is known
(hypothetical protein XP_044953.1; E=5e-9 and unnamed protein
product AK022687; E=1e-12, respectively), and are expressed in
neurons of both the planarian central and peripheral nervous system (see
http://planaria.neuro.utah.edu).
Our previous demonstration that double-stranded RNA can be used to inhibit
gene expression in planarians
(Sánchez Alvarado and Newmark,
1999) provides the means for testing gene function on a large
scale, thus allowing the functional characterization of novel, evolutionarily
conserved gene products.
Furthermore, cell type-specific markers identified by large-scale in situ
screens provide useful reagents for examining the processes of patterning,
differentiation and remodeling in intact and regenerating planarians. We have
shown the use of such a marker (H.112.3c) to quantify cell number changes as
planarians alter their size, and found that these animals also regulate
accordingly the numbers of a specific cell type
(Fig. 3). This maintenance of
pattern and proportion is a fascinating corollary to the regenerative
abilities displayed by these organisms. In addition, little is known about the
heterogeneity of the stem cell population in planarians and markers such as
piwi (H.2.12c) will provide necessary reagents for analyzing the processes by
which neoblasts differentiate to give rise to the 30 cell types in the
animal. The tools described make these daunting problems more amenable to
molecular dissection.
Finally, BLASTn and BLASTx queries also revealed that 31% of the cDNAs
obtained do not share sequence similarities with the available databases. This
lack of similarities with GenBank and dbEST is not due to the divergences
commonly found in untranslated sequences, because only
20% of these cDNAs
lack a putative ORF. These results suggest that some of these sequences may
correspond to Platyhelminth-specific genes. Therefore, in addition to its
obvious advantages for studying the problem of regeneration, the easily
manipulable planarian provides a free-living counterpart likely to complement
current research efforts on the parasitic forms, in particular Schistosoma
mansoni and S. japonicum, for which abundant sequence data are
being obtained (Snyder et al.,
2001
). Given that the parasitic flatworms are difficult
experimental subjects, the ability to identify flatworm-specific genes through
comparisons to S. mediterranea sequences should help identify
candidate molecules for therapeutic intervention. Furthermore, the in situ
hybridization data being generated in S. mediterranea will help
identify genes expressed in cell types unique to the platyhelminthes,
providing additional potential therapeutic targets. The combination of
sequence comparisons, gene expression patterns, and RNAi technology provide
new experimental possibilities for studying the free-living and parasitic
members of this phylum. Thus, the SmedDb resources will be useful to a wide
gamut of developmental and biomedical endeavors.
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ACKNOWLEDGMENTS |
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Footnotes |
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