Tardigrades: A new model organism
Developmental biology of tardigrades
Blaxter lab, ICAPB, Edinburgh
below is the text of the grant submitted to the BBSRC for funding
Tardigrades, also known as water bears, were first described 200 years ago. They are small animals, inhabiting marine, fresh water and water film habitats, with herbivorous, fungivirous and carnivorous feeding habits (Nelson, Higgins 1990). We here propose to ustilise tardigrades as a new model for examining the evolution of core developmental processes such as axis formation and appendage evolution using a combination of genomic, molecular genetic and developmental biology approaches.
Tardigrades are placed in a phylum, Tardigrada, that has traditionally been allied with the Arthropoda. Several authors have however placed Tardigrada within the Aschelminthes, a diverse (and probably paraphyletic) supertaxon comprising many "pseudocoelomate" phyla, including the Nematoda. Features that strongly suggest that they are related to arthropods are the possession of legs, and a distinctly segmented body (Brusca, Brusca 1990; Nielsen 1995), but they also have a nematode-like triradiate pharynx with stylets (seen in several groups of nematodes). Are tardigrades nematodes with legs, or arthropods with a pharynx?
Recently, molecular phylogenetic analysis suggested that tardigrades are related to both nematodes and arthropods in a new grouping, the "Ecdysozoa" (moulting animals) (Aguinaldo, et al. 1997). The Ecdysozoa brings together the two foremost invertebrate model organisms, Drosophila melanogaster and Caenorhabditis elegans, suggesting that the evolutionary history of these two diverse animals might include the acquisition of extremely divergent modes of development. For example, the Drosophila body plan is patterned in part by a set of genes, the homeobox cluster (HOX) genes, that have roles in anterior-posterior pattern element determination (Akam 2000). Related genes play similar roles in vertebrates: these genes are part of the basic process of development in all metazoans (Akam 1995). The C. elegans HOX cluster lacks some orthologous HOX gene groups, does not display spatial colinearity and has divergent sequences compared to all other phyla. The roles these genes play in patterning C. elegans are much more subtle than in flies and vertebrates, attuned to a highly determined mode of development (Costa, et al. 1988; Kenyon 1994; Salser, Kenyon 1994; Wang, et al. 1993).
The new taxonomy suggests that the C. elegans cluster may be significantly derived or degenerate: it may represent a pinnacle of simplification of HOX gene roles in patterning, or a "cluster" that is no longer intimately involved in A-P patterning. HOX sequences are available for a few taxa in the "Ecdysozoa" (de Rosa, et al. 1999). We are generating HOX datasets from other nematodes to investigate whether the C. elegans pattern is representative of all nematodes. Already we can say that while some of the C. elegans genes are indeed apparently nematode-specific, there is also evidence for additional HOX genes in other nematodes, ie loss of genes in the model nematode (Aboobaker and Blaxter, unpublished).
While many other facets of development can be compared between fly and worm, these two model organisms appear to be derived representatives of their respective phyla (Phillipe, et al. 1994). Comparison between "the worm" and "the fly" may be deeply uninformative (analogous to a line drawn between two outlying datapoints). In this context we would like to examine as a "linking" group, the tardigrades (also known as "water bears", phylum Tardigrada) (Kinchin 1994; Morgan, King 1976; Nelson, Higgins 1990; Nelson, Marley 2000). We would like to begin by describing the basic early embryology of tardigrades, develop genomic resources for tardigrades, clone and sequence a select set of developmental genes from tardigrades and investigate their expression in early development.
The Ecdysozoa hypothesis (Aguinaldo, et al 1997) has had a far reaching effect on invertebrate biology, suggesting as it does that the two "model" invertebrates are more closely related to each other than to other non-ecdysozoan phyla. A clarification of the differences and similarities between nematodes and arthropods would be of utility in both universalising the lessons learned (and to be learnt) from the fly and worm projects and in itemising the peculiarities of both systems. The tardigrades are an ideal linking group, and thus fit well into the Comparative Development programme call for proposals. In particular we hope to begin to be able to define how differences in gene sequence, expression and action result in the extreme differences in form seen between Ecdysozoa phyla.
We will lay the foundation for much future comparative work by providing at the outset a body of basic developmental, genomic and molecular genetic data for others to exploit. By combining the expertise of a nematode/genomics/phylogenetics group (Blaxter, Edinburgh) and an arthropod/development/evolution group (Akam, Cambridge) we will fuse several organismal and disciplinary fields, yielding a productive and stimulating synthesis. As far as we are aware there is no significant activity on tardigrade molecular developmental biology outside the UK, and this project would be a major first step in establishing a new research direction in UK developmental biology.
We have proposed work addressing only a very few of the possible approaches and systems applicable to tardigrades. Their ability to undergo cryptobiosis (living for up to 100 years in an anhydrobiotic state), the development of the nervous system, the homology between structures and development in the nematode and tardigrade pharynx, the comparative development of limbs in flies and tardigrades and the biology of the adult moulting clock are some of the future possibilities of relevance to both basic evolutionary developmental biology and applied science.
We have established in monoxenic culture a single, herbivorous tardigrade species, currently identified as a member of the genus Isohypsibius. The culturing of this animal is important: tardigrades are reputed to be unculturable, and there are three scattered descriptions of cultures in the literature, none still extant (Nelson, Marley 2000). The system is monoxenic (involving co-culture with clonal Chlamydomonas algae) and robust. As the species is self-fertilising (probably hermaphroditic), single tardigrades can be cultured in isolation, and large quantities can be generated free of substrate and food source contamination. We have developed methods for observation of tardigrade eggs under DIC microscopy, and have isolated ample DNA and RNA for molecular biology. We have already cloned small subunit ribosomal RNA genes and several HOX gene fragments for phylogenetic and evolutionary biology of development analyses (Aboobaker, Welsh and Blaxter, unpublished).
See here for a further defence of the "intermediate" or "linking" phylum status of tardigrades.
Outline of work
1 Description of the embryology and lifecycle of Isohypsibius sp. ED using in vitro culture, live DIC microscopy, histochemistry and fluorescent dye tracking fate mapping.
An embryo of
Isohypsibius sp. developing withing the eggshell. The
eggs are laid in the shed cuticle. The eggs are ~100 microns
long.
Tardigrade developmental biology is an understudied field (Nelson 1982) with only the basics of development described in a few taxa. Tardigrades are reported to be eutelic, and develop directly (rather than going through metamorphosis). Adult tardigrades continue to moult, often coincident with egglaying. We have established the basics of the lifecycle of Isohypsibius sp. ED in the laboratory. We can culture organisms in bulk, in aerated coculture with Chlamydomonas reinhardti algae as food source, and rapidly amass large numbers. Individual tardigrades can be picked to single wells of microtitre plates and cultured through several moults. As the eggs are laid within the moulted cuticle, we can also track synchronously developing eggs from individual animals. Individual eggs (or sets of eggs bound in a cuticle) have been cultured to hatching (which takes ~5 days at 20°C), and the hatchlings cultured to egglaying, a process that takes about 10 days. The animals appear to be self-fertilising (the genus Isohypsibius includes several known hermaphrodites), and single animals can be cultured to yield hundreds of progeny of several generations in small volume dishes. Cultures can be maintained in small volumes through the use of high Stokes ratio silicon oil overlays to prevent evaporation. We do not yet know when and if sex ever happens in this species. The eggs are readily handled and viewed under DIC microscopy, and are transparent. All stages, from egglaying through early cleavages, morphogenesis and hatching have been observed under DIC microscopy. Individual adults lay from one to nineteen eggs per shed cuticle, and moult about once a week. Egg supply is thus not problematic, and we are investigating methods of egg purification from mixed culture. We are investigating cryptobiotic and cryopreservation of stocks.
A late-stage embryo at
the completion of morphogenesis. Through the eggshaell, the
pharynx and the claws on the end of each of the eight legs
can be seen. Micrograph dfrom Bob Goldstein, UNC Chapel
Hill.
We propose to use highpower DIC and confocal microscopy to examine and record the early embryogenesis of mounted animals. Our goal will be to answer the questions
what are the major milestones of tardigrade development and how do these compare with D. melanogaster (and other arthropods) and C. elegans (and other nematodes)?what cellular and tissue structures (such as the primordia of segments and the pharynx) are laid down when in tardigrade development?
how uniform is development between individuals?
can we construct a fate map for tardigrade embryos, and how does this compare with maps of fly and worm?
do tardigrades have a strongly determined cell lineage (like nematodes) or are they regulative (like arthropods)?
are tardigrades eutelic (with constant cell number, like nematodes)?
Multiple animals will be followed through embryogenesis using timelapse videomicroscopy. Digital images will be annotated and compared between embryos. Staged embryos will be stained with bis-benzimide and viewed under fluorescence to count nuclei and time nuclear divisions. Hatchlings, young and older adults will also be stained and their nuclei photographed and counted. Standard histological staining will be carried out and sectioned animals photographed and the anatomy described. Fluorlabelled probes such as TRITC-phalloidin and fluorescinated lectins will be used to test methods of permeabilising embryos while maintaining anatomical integrity. Immunocytochemical staining using antisera to highly conserved antigens will reveal patterns of localisation of gene expression. Stained tardigrades will be imaged at high resolution using the ICAPB confocal laser scanning microscope facility. Microinjection of high molecular weight dyes and fluor-tagged molecules will be used to examine blastomere integrity and communication during development, and to track cell fates in hatchlings. As many techniques have been proven in other systems, we will focus on technology transfer from the fly and worm fields to tardigrades.
2 Preparation and arraying of a large insert genomic DNA library and representative cDNA libraries from Isohypsibius sp. ED.
In order to facilitate analysis of Isohypsibius sp. ED we would like to generate cloned DNA resources at the outset of the project. These resources will be central to many of the directed approaches to be taken later. We will first estimate the DNA content (size) of the Isohypsibius sp. ED genome by fluorescent dye staining of nuclei, and reassociation kinetic analysis of isolated DNA: tardigrade genomes are estimated to be in the order of 120 Mb. High molecular weight genomic DNA will be prepared from tardigrades. The DNA will be cleaved with appropriate restriction enzymes and two large-insert genomic DNA libraries made, one in a cosmid vector (superCos, ~30 kb inserts) and the other in a bacterial artificial chromosome vector (pBACe3.6, ~100 kb inserts). We have experience of genomic library construction in Edinburgh from the nematode genomics program (Williams, et al. 2000) The libraries will be verified by hybridisation screening with (a) bulk Isohypsibius sp. ED DNA (to identify repetitive sequence containing clones) and (b) probes derived from cloned genes of Isohypsibius sp. ED (ribosomal RNA, cDNAs, HOX PCR fragments).
Poly(A)-containing messenger RNA will be purified from tardigrades and used to construct cDNA libraries. We will construct a mixed-stage and an egg library at the outset. The libraries will be constucted in the phage vector lambdaZapII, and in the plasmid pSPORT. The libraries will be verified by insert sizing and test sequencing of 200 random clones, following established lab protocols (Daub, et al. 2000).
3 Expressed sequence tag analysis of cDNA libraries and genome survey sequence analysis of the large insert genomic DNA library.
To understand the biology of Isohypsibius sp. ED in more detail, we would like to examine the genes it expresses, and perform a preliminary survey of the genome. These studies will yield reagents useful for a number of downstream projects:
an estimate of the base composition of coding and noncoding tardigrade DNAan estimate of gene density in the tardigrade genome, and of gene number
an estimate of codon usage and mRNA structure in tardigrades
a survey of gene diversity in the tardigrade genome
a set of housekeeping genes to use as hybridisation and RT-PCR controls
a set of genes that may be useful subjects of downstream experiments in phylogenetics, developmental biology, transgenesis and RNAi.
We will pick 10,000 random cDNA clones to microtitre plates, and use high throughput sequencing methodologies to generate 5,000 expressed sequence tags from these. The ESTs will be analysed using a custom suite of software scripts devised in Edinburgh for analysis of nematode and other datasets (see http://www.nematodes.org) (Daub, et al. 2000; Williams, et al. 2000). These include clustering by sequence identity into EST groups deriving from single genes, and similarity analyses of the potential encoded proteins. The sequences will be deposited in the public database dbEST.
We will end-sequence 2,000 large insert clones from the BAC and cosmid libraries. Again, 10,000 clones of each library will be picked to microtitre plates and archived. For 2,000 clones, minipreparations of DNA will be made using a 96 well format method, and end sequence obtained using universal vector primers. The 4,000 end sequences (or genome survey sequences) will be analysed and annotated as above, and deposited in the public database dbGSS. The GSS (and perhaps the ESTs) may also provide us with evidence of possible retroelements (transposons and retrotransposons) that may be useful in future transgenesis experiments.The clones, sequences and analyses will be freely available from Edinburgh, through a project world wide web site, as is currently the case for our nematode genomics program.
4 DNA cloning, sequencing and recombinant protein expression for antibody production of selected developmental regulatory genes from Isohypsibius sp. ED.
In order to initiate molecular genetic analyses of development in the tardigrades, we have already cloned a six HOX gene fragments from Isohypsibius sp. ED using reverse transcriptase-PCR (Aboobaker, Welsh and Blaxter, unpublished). These include probable orthologues of HOX3, sex-combs reduced or deformed, Abd-b, labial and proboscipedia, and the para-Hox cluster gene caudal. Ftz has been cloned from another tardigrade species (Telford 2000). We propose (a) to complete the HOX gene set for Isohypsibius sp. ED, and obtain full length cDNAs for all the HOX cluster genes and (b) to clone and sequence cDNAs for additional sets of developmental regulatory genes. The list of possible candidates is long, but we feel we can easily devise cross-phylum primer sets for the TGF beta / bone morphogenetic protein / decapentaplegic family of ligands and receptors and the Toll receptor family. These would allow us to look at conservation of the dorsal-ventral patterning system, a major difference between development of C. elegans and D. melanogaster. Another primary target for this approach will be the PAX-6 gene, involved in eye determination (Isohypsibius sp. ED in common with most tardigrades has paired anterior eyespots), and tinman/NK-2/ceh-22 involved in heart/pharynx patterning. The latter two genes should be readily amenable to PCR cloning as they are divergent, but conserved, homeodomain proteins.
Degenerate primer sets will be designed based on alignments of gene families and used to screen cDNA and genomic DNA preparations. The cDNAs will be derived from both mixed stage and selected embryo material. Partial cDNAs will be verified by sequencing and extended 5 and 3 using RACE technologies. Full length sequences will be compared with orthologues form other phyla to examine patterns of amino acid conservation and substitution, and the phylogenetic signal present in these developmental control genes investigated in the light of our nematode HOX data and the extensive arthropod HOX data.
5 In situ and immunohistochemical analysis of expression of developmental regulatory genes in Isohypsibius sp. ED embryos.
We will develop an in situ hybridisation method for Isohypsibius sp. ED. This will be based on that developed for arthropods and C. elegans. Digoxygenin labelled cDNA probes will be hybridised to fixed and permeabilised embryos and visualised by enzymatic staining. Embryos will be counterstained to identify labelled structures. In situ hybridisation on sectioned embryos will also be used to reveal detailed anatomical location. We will also attempt the use of fluorescent antibody-anti-digoxygenin and fluor-labelled probes and confocal visualisation.
Using this method, the expression of the HOX genes, the TGFbeta family genes and the PAX and tinman orthologues will be followed through embryogenesis. Staged embryos can be isolated from single adults maintained in isolation, where the time of egglaying and moulting can be ascertained with some accuracy.
As an example of the kind of data we expect to be able to generate, consider the limbs of tardigrades. Tardigrades have four sets of paired limbs, the posterior pair being directed posteriorly and little used for locomotion. These limbs correspond with apparent segmental pattening of the body. There are no head appendages (a few genera have circumoral sensory papillae that do not have the morphology of appendages), and the head is distinguished by the presence of a nematode-like pharynx. Are tardigrade legs and segments homologous to arthropod legs/segments, and if so, which legs/segments are they homologous to? We will prepare labelled probes corresponding to the segmental patterning HOX genes of arthropods and perform in situ analysis on embryos and emergent larval tardigrades. The pattern of HOX gene expression in the tardigrades will permit allocation of appendages and segments to putative homology groups. Similar questions as to eye development (PAX-6 gene) and pharyngeal/heart development (ceh-22/tinman gene) will also be asked. The TGFbeta pathway plays a core role in alternate development and diapause in C. elegans: is the same true of tardigrade TGFbetas and cryptobiosis?
6 Preliminary analysis of gene function in Isohypsibius sp. ED using RNA mediated interference and green fluorescent protein expression transgenesis techniques.
Double stranded RNA corresponding to expressed exons of genes has a significant inhibitory effect on expression of the corresponding genes in many species (Baker, Macagno 2000; Bastin, et al. 2000; Bosher, Labouesse 2000; Caplen, et al. 2000; Fire, et al. 1998; Fraser, et al. 2000; Hughes, Kaufman 2000; Lohmann, et al. 1999). RNA interference is mediated through catalytic degrading of mRNAs and results in phenotypic knock-out or knock-down of gene expression. RNAi is a core method in functional genetic analysis for many organisms.
For a set of genes for which we expect to be able to detect a phenotype (for example presumably essential housekeeping genes, and regulated expression developmental genes) we will make double stranded RNA by recloning the cDNAs in a commercial vector having two T7 polymerase sites flanking the cloned insert. dsRNA will be synthesised in vitro using T7 polymerase, and tardigrades exposed initially by soaking in small volumes of dsRNA at high concentration. We will monitor RNA entry into the tardigrades by labelling RNA with fluor-tagged nucleotides. If dsRNA soaking does not yield results, we will proceed to dsRNA microinjection of embryos.
The ability to construct transgenic animals has been a mainstay of developmental biolgy. We would like to pilot transgenesis in the tardigrades by preliminary experiments using inducible and constitutive promoters driving both green fluorescent protein and beta-galactosidase reporter genes (derived from the Fire laboratory vector library (Fire 1986)). If successful, this technique will allow us to examine the temporal and spatial expression paterns driven by gene promoters in live and fixed animals.
cDNAs for heat shock proteins (large and small), and for costitutively transcribed genes (for example tubulin, or ribosomal protein genes) will be selected from the EST dataset. These cDNAs will be used to probe the genomic DNA libraries (gridded at high density from the 10,000 clones per library picked) for genomic copies of the respective genes. The positive BAC or cosmid clones will be restriction mapped and fragments from the 5 upstream and 3 downstream regions of the selected genes subcloned and sequenced. From these sequenced fragments, putative control regions (say the 2-5 kb upstream of an ATG start codon) will be subcloned into the Andy Fire beta-galactosidase-GFP vector pPD series (Fire 1996). As the presence of introns and a competent set of 3 signals for polyA addition and transcription termination are important for expression in other systems, we will substitute the C. elegans derived signals in the pPD vectors with presumptive signals derived from the Isohypsibius hsp genes.
Purified plasmid DNA will be microinjected under DIC optics into adult gonads and into embryonating eggs. In order to follow possible transgenic animals we will also coinject nondiffusible dye-labelled dextran (or other cell-autonomous marker found to be useful in the developmental biology studies in 1 above). Potential transgenic animals will be subjected to heat shock, and viewed under fluorescence epiillumination. Once successful microinjection transformation is achieved, the functional vector system will be used to examine promoter function in selected developmental control genes.
7 Phylogenetic comparison of developmental patterns and regulatory gene evolution (sequence and function) between Isohypsibius sp. ED and other organisms, particularly members of the Ecdysozoa.
We have assessed the phylogenetic position of Isohypsibius sp ED using the nuclear small subunit ribosomal RNA gene (Welsh, Aboobaker and Blaxter, unpublished), and have confirmed its identification in comparison to the other eleven tardigrade SSU sequences available. To confirm and extend this analysis, in particular with a view to using tardigrade to clarify phylogenetic analysis of development in the Ecdysozoa, we will sequence in full additional phylogenetically informative genes, using the EST dataset as a source of clones. Probable subjects of this effort will include elongation factor 1-alpha and the large subunit of RNA polymerase II (sequences available for another tardigrade, Milnesium sp.), and the complete mitochondrion. Phylogenetic analysis using gene order data (mitochondrial) and nucleotide and amino acid alignments will be used to derive robust hypotheses of the relationship of Isohypsibius sp. ED to other tardigrades and ecdysozoans using all available methods (Blaxter, et al. 1998; Swofford 1999; Swofford, et al. 1996). These hypotheses will allow us to map and examine the patterns and dynamics of evolutionary change in developmental gene expression in the ecdysozoa.
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