Tag Archives: Lamprey

Genomic Rearrangement in Lampreys 2

As discussed in a recent post, during lamprey embryogenesis programmed genomic rearrangements lead to deletion of ~20% of the germline genome in the soma. Smith, Amemiya and co-workers have now published a follow-up study in which they further characterise the complement of deleted genes. Their findings have led them to hypothesise that the programmed genomic rearrangements (PGRs) serve to segregate pluripotency functions required in germline that could be deleterious in the soma.

Smith et al used a couple of different genomic techniques to identify somatically deleted sequences. Using microarrays constructed from available germline sequence, they found that ~13 % of the sampled sequence was deleted in the soma (in relative agreement with the ~20% derived from flow cytometry). Within this dataset, they identified 8 new single-copy/low-copy number sequences found only in the germline. RT-PCR showed that 5 of the novel sequences were expressed in germline cells. In situ hybridisation of one of these sequences showed that it was expressed in differentiating primordial germ cells in lamprey embryos.

The main limitation for identifying more genes subject to somatic deletion has been a lack of germline genomic sequence. Smith et al. performed high-throughput shotgun sequencing on lamprey sperm cells, generating short sequence reads covering ~10% of the germline genome. They then compared this dataset with the whole-genome sequence derived from somatic (liver) cells, yielding tens of thousands of putative deletion and recombination sites. A substantial part of the somatically deleted DNA corresponds to single-copy, protein-coding genes; the authors identified 246 instances of homology to individual human genes.

The problem with this comparison however, is that, by necessity, it was generated from 2 different individuals (of different sexes). This meant that apparent cases of deletion or recombination may be due to polymorphisms for insertion or deletion mutations present in lamprey populations. The researchers undertook validation experiments on a subset of the candidate deletion/recombination dataset (using PCR to amplify candidate sequences from testes and blood from 4 different males, blood from 4 different females, as well within an array of somatic tissue types within individuals). Of 48 tested candidate gene deletions or recombination events, they validated 7 sites of programmed deletion, and 3 recombination sites. They also identified 3 insertion/deletion polymorphisms, and 5 gaps in the somatic whole genome sequence. Due to PCR failures, or because of repetitive target sequences, 30 of the candidates were not informative. The validated gene deletions included APOBEC-1 complementation factor, encoding a protein involved in RNA editing, and the secreted developmental signalling molecule encoding WNT7A/B.

In the process of these validation experiments, Smith et al discovered short palindromic sequences at the deletion breakpoints. There was no specific consensus sequence at these positions, but the palindromes may indicate that the mechanism of chromatin diminution utilises site-specific recombination.

Another interesting finding of these experiments is that it appears that the programmed deletions are inherited uniformly throughout all the various somatic lineages. The earlier paper (discussed in the previous post) had suggested that different somatic tissues might have subtly different deleted portions. Microarray experiments, and comparison of the validated gene deletions between different tissues found no evidence of this, although this question may as yet not be answered definitively.

The crux of the paper rests on a computational comparison of ontology terms (in which homology is used to make predictions of cellular function, which are further sorted into broad categories). In the dataset of predicted gene deletions, certain ontologies were overrepresented with respect to the rest of the germline sequence; these included ‘regulation of gene expression’, ‘chromatin organisation’, and ‘development of germ/stem cells’.

Simply put, the paper has shown that a substantial number of protein-coding genes as well as repetitive sequences are deleted from the genomes of lamprey somatic cells. Many of the deleted genes are expressed in the germline, and often appear to have important regulatory functions. The crucial characteristics of the germline are the ability to undergo meiotic recombination, and totipotency. The missexpression of factors involved in these processes in the soma would be seriously detrimental; potentially resulting in aberrant cell fate specification, genome disruption, and hence cancers. The authors postulate that this conflict of interests between the germline and the soma underlies their genomic differentiation.

I find this an attractive and interesting hypothesis. As yet though, I don’t think the data is strong enough to have proved it. Gene ontology terms are relatively crude categorisations, and compounded with the question of what proportion of candidate deletions are bona fide, I’ll withhold judgement on the evolutionary rationale behind the deletions for the moment. Jeramiah Smith and colleagues are currently assembling the entire lamprey germline genome. Complete annotation of the deleted portion of the genome will certainly reveal the function of these fascinating genome rearrangements more clearly. I look forward to new studies investigating the mechanisms underlying the rearrangements, and their developmental progression. The extensive genome remodelling that occurs in ciliates utilises a combination of a small RNA/Argonaute system and domesticated transposase enzymes. I guess that analysis of any transposases encoded in the lamprey genome may be the place to start to unravel the mechanisms of chromatin diminution.

Smith JJ, Baker C, Eichler EE, & Amemiya CT (2012). Genetic consequences of programmed genome rearrangement. Current biology : CB, 22 (16), 1524-9 PMID: 22818913

Genomic Rearrangement in Lampreys

Generally, all cells in an organism are considered to have the same genome; the differences between cells being determined by differential expression of genes. However, a growing list of species, including sciarid flies, and various copepods and nematodes, are known to undergo genomic rearrangements during the differentiation of cell lineages. Recent findings have shown that two species of jawless vertebrates (cyclostomes), the hagfish and the sea lamprey, undergo extensive genomic remodelling during development.

Hagfish and lampreys are the closest extant relatives of the jawed vertebrates (gnathostomes). Controversy has reigned over whether lampreys are more closely related to gnathostomes than they are to hagfish. This debate appears to have been resolved by molecular phylogenetics; the two branches of jawless vertebrates are united as a monophyletic clade, the cyclostomes. However the division of hagfish and lampreys occurred shortly after the division of gnathostomes and cyclostomes in the early stages of vertebrate evolution (~500mya). Two whole genome duplications occurred during the early evolution of the vertebrates (referred to as 1R and 2R). It is not yet resolved whether these duplications occurred before the divergence of cyclostomes and gnathostomes. Opinion appears to be split as to whether this divergence occurred after 1R or after 2R. The easiest way to resolve this question is whole genome sequencing. It has been known for some time that hagfish undergo genomic rearrangements during early development. Smith et al (2009) have now shown a similar phenomenon occurring in the Sea Lamprey (Petromyzon marinus), explaing some of the difficulty in constructing a finished version of the sea lamprey genome.

Smith et al. found that the total DNA content of nuclei from germline (sperm) and various somatic cell types (blood, liver, kidney) differed by >20%, equating to ≈500Mb. They then performed southern blots in which the restricted genomes of blood or sperm cells were probed with a repetitive sequence element. A number of bands differed in size or intensity between the germline and somatic cells, showing that genome rearrangement had occurred. One specific band was present in the germline samples and virtually absent from a range of somatic tissues. This band, termed Germ1 consisted of sequences for the 18s ribosomal DNA, a retrotransposon, and a section of the 28s rDNA. When aligned to Lamprey genomic sequence (somatic cell derived), these sequences were all commonly found, but one section of Germ1, from one end of the fragment to the 28s rDNA section, was dramatically underrepresented; a germline specific sequence. Smith et al. then performed FISH (fluorescent in situ hybridisation) using the Germ1 clone against metaphase germline and somatic cells. In the germline they found many Germ1-like sequences distributed across several chromosomes, often arrayed in tandem repeats. In contrast, in somatic cell nuclei, Germ1 hybridised to only one chromosome pair, most likely equating to the functional rDNAs. Using real-time PCR over the period of early embryogenesis, the authors found Germ1 abundance was drastically reduced 2 or 3 days after fertilisation. Smith et al. estimate that Germ1-like sequences make up ~7% of the germline genome, therefore suggesting that ~13% more of it is also lost. By comparing germline BAC clones with somatic genome sequence, they managed to identify more lost sequences including a gene known to be expressed during germline development, SPOPL.

Smith et al. have therefore shown that the sea lamprey genome undergoes a dramatic rearrangement during the early development of the somatic tissues. A large proportion of the excised content is accounted for by the elimination of Germ1-like sequences. However, it appears likely that a number of specific genes are also lost. In most cases of large scale somatic genomic rearrangement the main excised component is made up of transposable elements and other repetitive sequences. It seems likely that this could be the main basis for that seen in lampreys, as Germ1-like sequences appear to be a strange fusion of transposon and duplicated ribosomal DNA genes. However, the finding that an individual gene, SPOPL, is also selectively deleted in somatic lineages suggests that genomic deletions could be linked to the genetic regulation of development.

Interestingly, when the common apoptosis assay TUNEL – which detects DNA breaks – is used during the first few weeks of lamprey embryonic development, nearly every nucleus is labelled. It seems likely that this effect (that was considered an artifact) is explained by developmentally regulated deletions. The deletion of SPOPL appeared to occur more gradually than that of Germ1- like sequences. Together with the observation that the total nuclear DNA content differed slightly between various somatic tissues, these findings suggest that this program of somatic genomic deletions could be occurring in an intricate, and tissue specific, progression during early development.

All jawed-vertebrates undergo programmed genomic rearrangements during the diversification of the immune system. VDJ recombination, mediated by the transposase derived RAG recombinase, generates antigenic diversity, allowing adaptive immune responses. Cyclostomes have an alternate RAG-independent adaptive immune system, termed VLR. The recombinational system used in VLR is not yet clear. Is this system linked to that employed during the early developmentally regulated deletion process?

Smith et al (2010) link the presence of chromatin diminution (ie the somatic genomic rearrangements) in this basal vertebrate taxon to the whole genome duplications that occurred in the vertebrate stem group. If the genomic rearrangement mechanisms seen in cyclostomes were present in the last common ancestor of jawed and jawless vertebrates perhaps this system predisposed the stem group vertebrates to whole genome duplications by creating a permissive environment for polyploidisation and rediploidisation? Although this is a fascinating idea, currently it is perhaps a slightly idle speculation. It is of pressing importance to understand the mechanisms underlying cyclostome genomic rearrangements. Are they the same between hagfish and lampreys? Are similar systems present in gnathostomes? Finished genome sequences for both germline and various somatic cell lineages will answer many questions regarding the effects and purposes of chromatin diminution, however one can understand that this is easier said than done considering the potential complexity of the rearrangements. Lampreys and hagfish are also so far unculturable in the laboratory, adding to the difficulty in expanding their experimental use. Perhaps the biggest question left hanging, is whether other jawed vertebrates employ programmed genomic rearrangements for purposes other than antigenic diversification? This remains a possibility as the consistency of the genome has been generally assumed rather than tested.

See also a follow up on a new paper from the same group: Genomic Rearrangement in Lampreys 2

Smith JJ, Antonacci F, Eichler EE, & Amemiya CT (2009). Programmed loss of millions of base pairs from a vertebrate genome. Proceedings of the National Academy of Sciences of the United States of America, 106 (27), 11212-7 PMID: 19561299

Smith JJ, Saha NR, & Amemiya CT (2010). Genome biology of the cyclostomes and insights into the evolutionary biology of vertebrate genomes. Integrative and comparative biology, 50 (1), 130-7 PMID: 21558194

Shimeld SM, & Donoghue PC (2012). Evolutionary crossroads in developmental biology: cyclostomes (lamprey and hagfish). Development (Cambridge, England), 139 (12), 2091-9 PMID: 22619386
This article is good for vertebrate phylogeny and cyclostome development. A free version here