Category Archives: Development

Breaking Neuronal Symmetry by Chromatin Memories

The asymmetric fates of two bilaterally symmetrical neurons are determined by a two-step activation program at a miRNA locus. Very low levels of transcription ‘prime’ the locus many cell generations before the final fate determination is imposed by a bilateral ‘boost’.

Animal nervous systems are generally bilaterally symmetrical anatomically, whilst displaying many functionally important left-right asymmetries. How is asymmetry imposed on a bilaterally symmetrical ground plan? The nematode C. elegans with its invariant cell lineage and tractable genetics offers a powerful model system in which to tackle this issue. Cochella and Hobert have published an elegant new paper describing how a distinct chromatin state at a microRNA locus serves as a molecular mark encoding a memory of a cell’s ancestry in an asymmetric lineage.  After many cell generations, this mark engenders a different response to terminal differentiation from its’ bilaterally symmetric partner cell.

The bilaterally symmetrical pair of gustatory neurons ASEL(eft) and ASER(ight) express different repertoires of chemoreceptors. This functional asymmetry is underpinned by the differential expression of two transcription factors. DIE-1 is expressed in ASEL and COG-1 in ASER. Together with a microRNA, lsy-6, which represses COG-1 expression, they form a bistable feedback loop responsible for determining the asymmetric fates of ASE neurons. Loss of any of these three factors results in conversion of one ASE to the other. However, the asymmetric expression of die-1 and cog-1 only occurs within the post-mitotic neurons themselves. How then is this asymmetry established?

A schematic representation of the features of asymmetric ASE specification. Note the original asymmetry in the lineages is determined at the 4 cell stage, tbx-37/38 expression in the great-granddaughters of ABa, and lsy-6 in the loop in ASEL.

A schematic representation of the features of asymmetric ASE specification. Note the original asymmetry in the lineages is determined at the 4 cell stage, tbx-37/38 expression in the great-granddaughters of ABa, and lsy-6 in the loop in ASEL.

The two ASEs are derived from different cell lineages that diverge at the 4-cell stage. The two daughters of the 2-cell stage blastomere AB, ABa and ABp, differentiate from each other due to signalling from one of the other 4-cell stage blastomeres to ABp. This signalling event represses the expression in the ABp lineage of a pair of redundant transcription factors, TBX-37 and TBX-38, which are transiently expressed in the 8 great-granddaughter cells derived from ABa. The expression of these TBX proteins is crucial to the asymmetric fate specification of ASE neurons as in tbx-37/38 double mutants ASEL is converted into ASER. However, TBX-37 and 38 are only expressed in the lineage giving rise to ASEL six cell generations before it’s birth. This large gap between the different stages of asymmetric ASE determination lead researchers to postulate the existence of a ‘memory mark’ linking TBX-37/38 action to the expression of the asymmetry defining feedback loop.

During this hiatus between TBX-37/38 expression and terminal ASE determination, the lineages giving rise to the two neurons become symmetric. A number of left/right pairs of neuronal precursors expressing the proneural gene hlh-14 develop from the two lineages, but only the pair of ASE mother cells express the ‘terminal selector’ transcription factor CHE-1. CHE-1 drives the expression of many ASE-expressed genes and activates expression of the asymmetric loop components, lsy-6, die-1, and cog-1. It is at this point that the TBX-37/38-dependent memory mark must integrate into the bilateral activity of CHE-1 generating the asymmetric expression of the loop components.

To try to discover the nature of the memory mark Cochella and Hobert performed a detailed analysis of the expression of the loop components. lsy-6 was suggested to act upstream of die-1 and cog-1 by genetic experiments, and the researchers found that it was the first of the loop components to be expressed.  It is expressed asymmetrically from the start in the ASEL mother cell. Deletion of lsy-6 results in conversion of ASEL to ASER. A construct of lsy-6 in combination with 932 bp of upstream sequence is able to rescue this effect, but sometimes leads to the conversion of ASER to ASEL. This suggested that the ‘upstream element’ construct drove ectopic expression in ASER as well as ASEL. Indeed, the upstream element contains CHE-1 binding motifs causing expression in both the ASE neurons. Cochella and Hobert therefore assayed other lsy-6 surrounding sequence for cis-regulatory information limiting its expression to ASEL. In fact, a construct including the upstream element, lsy-6, and 300 bp of downstream sequence completely rescued lsy-6 null alleles, eliminated ectopic ASER conversion, and was expressed identically to the endogenous miRNA. Normal lsy-6 expression is therefore regulated by both the upstream and downstream elements.

When the downstream element was used alone to drive expression of a reporter gene, it produced a very different pattern. Expression started early in a few ABa-derived blastomeres, one cell division after the expression of tbx-37/38, continuing in the progenitive lineage of ASEL until its’ birth.It never drove expression in ABp derived lineages. Expression from the downstream element was completely lost in tbx-37/38 double mutants, whilst mis-expression of TBX-37/38 in ABp derived cells lead to ectopic expression of the downstream element reporter. The downstream element contains a predicted binding-site for T-box proteins, directly linking the lineage–dependent expression of tbx-37/38 with theasymmetry-defining loop.

The expression pattern driven by the downstream reporter suggested that lsy-6 may be expressed far earlier than previously observed. The researchers therefore used a very sensitive technique to image potential lsy-6 transcripts. This showed that a few lsy-6 RNAs were present in cells in the lineage giving rise to ASEL five generations before strong expression is observed in the ASEL mother cell.

Broadly therefore, lsy-6 expression occurs in two phases; a very low level of activation early, dependent on the downstream element, and a second upstream element-dependent higher level of expression in the ASEL mother cell. However, deletion of the downstream element within large genomic constructs abrogated expression at all stages, and failed to rescue lsy-6 null alleles. This contrasted with earlier observations in which the upstream element alone could drive expression and rescue. The difference between these observations suggested  that, within a normal genomic context, the upstream element can only function in combination with the downstream element.

The authors therefore posited a model in which early downstream element/tbx-37/38- dependent transcription may ‘prime’ the locus in some way, rendering it competent to respond to the later transcriptional ‘boost’ mediated by CHE-1 acting on the upstream element.

Cochella and Hobert tested their model by substituting priming via tbx-37/38/downstream element for priming via ectopic CHE-1. In worms with the downstream element deleted, they drove early expression of CHE-1 from a heat-shock promoter approximately 4 cell generations before its’ normal time of expression. This caused low levels of lsy-6 transcription, rescuing the priming phase and allowing later ASEL expression and determination. Priming is therefore not dependent on a specific transcription factor acting on the downstream element, rather as long as low levels of transcription occur at the locus, it is primed.

This suggested that the memory mark causing the different response of the lsy-6 locus may be a lineage-specific transcription-dependent chromatin state. Using a cunning technique to visualise the level of chromatin compaction on transgenic arrays containing the lsy-6 locus, they observed chromatin decompaction of thelocus in the ASEL progenitive lineage 1 cell division after tbx-37/38 expression. Chromatin decompaction is associated with active genes; in the absence of early transcription the locus becomes compacted and refractory to CHE-1 activation later. In tbx-37/38 double mutants this lineage-specific decompaction was never observed, nor was it seen when the downstream element was deleted.

The memory mark is therefore chromatin decompaction at a miRNA locus linked to very low levels of transcription imposed within a cell lineage at an early stage of development. This primed state relays asymmetric information into an otherwise bilaterally symmetrical developmental program, facilitating diversification of neuronal cell fates. The timing of the priming mechanism fits in with earlier evidence that C. elegans embryos are relatively developmentally plastic until the 64-128 cell stage when developmental genes become compacted and refractory to ectopic activation.

Although I find this paper very elegant and convincing, I do have a few qualms about the most crucial experiment: the early ectopic activation by CHE-1. It seems like a slightly dirty experiment and I think I would’ve preferred to see ectopic induction of lsy-6 transcription via an unrelated mechanism. Perhaps experiments such as these would’ve had their own problems and my doubts are unfounded. I would also have liked to see the compaction assay performed with the ectopic CHE-1 induced activation.

The demonstration of a chromatin-based lineage specific prepattern facilitating differential responses to more generic inputs later in embryogenesis has wide implications, not just for asymmetries in the worm nervous system, but for the way we understand development in many animals. Firstly a technical point; to visualise early lsy-6 transcription the authors had to use a very labour intensive and hi-tech form of in situ hybridisation. The transcription they found, of just a few individual RNA molecules per cell, had massive developmental significance. Generally the techniques used to judge expression in developmental studies is nowhere near as sensitive, implying that we may be missing a lot of important information. Secondly, a more general point; A cell or tissues’ ‘competence’ to respond to developmental signalling, a concept derived from experimental embryology, and perhaps disdained in more genetical perspectives is relevant here. Molecular memories encoded by chromatin states may be a very widespread mode for imposing pre-pattern or developmental competence during embryogenesis. It seems to me that these types of understandings can begin to blend together the two different meanings of epigenetics; namely the derivation of the word by Waddington from epigenesis (meaning the increase in complexity during development), with the more current usage of epigenetics as describing a diverse collection of non-genetic inherited information.

Cochella, L., & Hobert, O. (2012). Embryonic Priming of a miRNA Locus Predetermines Postmitotic Neuronal Left/Right Asymmetry in C. elegans Cell, 151 (6), 1229-1242 DOI: 10.1016/j.cell.2012.10.049

Uploading piRNAs to the Cloud.

A new paper finds a protein linking piRNA transcription with processing in nuage.

The Piwi/piRNA system is responsible for protecting the germline from the mutagenic effects of transposon mobilisation. As summarised in an earlier post, in Drosophila large arrays of transposon fragments, located in pericentromeric and subtelomeric chromatin domains give rise to long piRNA cluster transcripts. These transcripts are then processed to produce the 23-30 nt piRNAs which, when complexed with Piwi-family argonaute proteins effect the post-transcriptional silencing of transposons. Although a more limited piRNA system functions in the somatic follicle cells surrounding the Drosophila egg chamber, the bulk of germline transposon silencing is performed by the system active in the germline siblings of the oocyte – the nurse cells. Here, dual-strand piRNA cluster transcripts are processed in the nuage, a perinuclear electron-dense cytoplasmic structure, where the ‘ping-pong’ system of reciprocal cutting and complexing between the Piwi proteins Aubergine (Aub) and Ago3 leads to piRNA amplification.

Nuage is a hallmark of germline cytoplasm in animals, and appears to be the site of both piRNA processing and transposon silencing. A hierarchy of proteins responsible for the assembly and function of nuage has been revealed by studies in Drosophila. Vasa, a DEAD-box RNA-dependent helicase protein, is required for the localisation of Tudor and other Tudor-domain-containing (Tdrd) proteins. These serve as a platform for the piRNA system, binding Aub and Ago3. Defects in many of these piRNA biogenesis components do not just lead to uncontrolled transposon activity; rather, they affect the asymmetric localisation of RNAs in the developing oocyte – a process by which developmental prepattern is organised. Zheng et al. discovered that weak mutations in the uap56 gene caused similar defects, suggesting a potential role in piRNA biogenesis.

UAP56 is another DEAD-box containing RNA-binding protein. It is ubiquitously expressed, localised in nuclei and has previously been shown to be involved in mRNA splicing and export. Zheng et al. found that in nurse cells it localises to discrete foci in the periphery of the nucleus. This was a similar pattern to that of Rhino (Rhi), a Heterochromatin Protein 1 variant previously shown to associate with piRNA clusters. Indeed, UAP56 and Rhino co-localised ~99% of the time in nurse cell nuclei.  Mutations in either uap56 or rhi caused a failure in the focal localisation of the other protein, showing their co-dependence.

When Vasa was imaged at the same time, it became apparent that it localised to foci in the nuage directly across the nuclear envelope from UAP-56-Rhi foci. Co-labelling with a nucleoporin showed that in fact UAP56-Rhi foci and Vasa foci directly abut nuclear pores from either side.

In the absence of functional UAP56 the nuage fails to assemble properly; Vasa, Aub and Ago3 all fail to localise. Similar effects are observed in rhi mutants, placing both UAP56 and Rhino upstream of Vasa as extrinsic factors necessary for nuage assembly. The uap56 mutants also fail to produce a large part of the proper complement of piRNAs leading to a consequent mobilisation of transposons. No effects on the level of genic mRNAs were detectable. Due to the failure of nuage assembly, the uap56 mutants also display germline DNA damage and the morphological defects caused by mislocalisation of asymmetric RNAs.

DEAD-box containing proteins act as ATP-dependent RNA clamps. As Rhino is known to associate with dual-strand piRNA clusters, Zhang et al postulated that UAP56 may be binding and stabilising nascent cluster transcripts. Indeed piRNA cluster transcripts could be co-immunoprecipitated with UAP56 and Vasa.

The data therefore suggests an attractive model in which cluster transcripts are passed across the nuclear pore between the two DEAD-box containing proteins, UAP56 and Vasa. The authors term this a nuclear pore spanning piRNA processing compartment. piRNA cluster transcripts must in some way be marked and specifically transported via the trans- nuclear pore compartment.

Running through this work as a consistent undertone are the implicit links to the broader RNA processing systems. The nuage is obviously intricately linked to the differential transportation of RNAs from the nurse cells and around the oocyte. UAP56 has other roles in mRNA splicing and export from the nucleus. What exactly are the links between the germline specific role of UAP56 and the general RNA splicing and export machinery? Zhang et al end with the enticing observation that mutations in two different genes encoding conserved exon junction splicing components also lead to similar asymmetric RNA localisation defects. It appears that the control of piRNA processing and transposon silencing in nuage is intimately linked to broader networks controlling germline specification and the patterning of the oocyte. Although the different strands of these systems are difficult to tease apart, Drosophila oogenesis continues to offer an unparalled paradigm for their investigation. The piRNA system is widely conserved in animals, but there does appear to be quite a lot of plasticity in its specifics. For instance, as discussed at length in this series of posts, in C. elegans, piRNAs are individually transcribed. I’d be very interested to find out whether homologues of Rhino and UAP56 play any role in this system? I’ll riff on the similarities and differences of piRNA systems and their links to development some more in future posts.

Zhang, F., Wang, J., Xu, J., Zhang, Z., Koppetsch, B., Schultz, N., Vreven, T., Meignin, C., Davis, I., Zamore, P., Weng, Z., & Theurkauf, W. (2012). UAP56 Couples piRNA Clusters to the Perinuclear Transposon Silencing Machinery Cell, 151 (4), 871-884 DOI: 10.1016/j.cell.2012.09.040

Lin, H. (2012). Capturing the Cloud: UAP56 in Nuage Assembly and Function Cell, 151 (4), 699-701 DOI: 10.1016/j.cell.2012.10.026

Epigenetic Licensing of a Sex Determination Gene

A recent series of papers describing interacting small RNA pathways in C. elegans, posited the existence of an anti-silencing pathway responsible for licensing the expression of germline transcripts. Johnson and Spence (Science. 2011) published the first paper suggesting such a pathway. Using an elegant series of genetic experiments, they found that maternally supplied RNA of the sex determination gene fem-1 was necessary for normal zygotic fem-1 activity. In the absence of this maternal component, normal fem-1 alleles are epigenetically silenced.

In C. elegans, sex is determined by the ratio of sex chromosomes to autosomes. Worms with one X chromosome become males, whilst those with two develop as self-fertile hermaphrodites. Mutations in genes in the sex determination pathway can give rise to true females. For instance, fem-1 is required for masculinization in both males and hermaphrodites; worms deficient for both maternal and zygotic fem-1 activity lack all male development.

Johnson and Spence noticed some discrepancies between the relative importance of the maternal and zygotic components of fem-1 action. Homozygous fem-1 mutants, born of heterozygous mothers show partial male development because of maternally supplied fem-1. Zygotically heterozygous progeny of female worms homozygous for point mutations in fem-1 are phenotypically normal; that is to say that despite the lack of any maternally derived active FEM-1 protein, male development can proceed normally. However, similar crosses, in which fem-1 deletion alleles are used rather than point mutations, give rise to heterozygous worms showing feminization of the germ line (Fog phenotypes). To prove the requirement of a maternal fem-1 product for spermatogenesis, Johnson and Spence performed a clever genetic trick that produced worms that were homozygous for the wild-type fem-1 allele but were born of a mother homozygous for the deletion mutation. Most of these worms also exhibited Fog phenotypes. Other strong loss of function alleles that eliminated FEM-1 protein, could complement (ie. rescue) the deletion mutation-caused maternal effect. It therefore appeared that maternally derived fem-1 RNA was required for spermatogenesis, independently of its protein-coding function.

To confirm this, the authors injected in vitro transcribed fem-1 RNA into females homozygous for the fem-1 deletion allele. These worm’s progeny displayed fewer Fog phenotypes than those of uninjected worms. A similar level of rescue was observed when fem-1 RNA without a translation initiation codon was injected, showing that FEM-1 protein was not required.

What was the function of the maternal fem-1 RNA? Johnson and Spence hypothesised that it might be required for normal zygotic fem-1 activity. They therefore assayed this in heterozygotic worms descended either from mothers homozygous for the deletion, or from heterozygotes. Both a genetic test and observation of zygotic fem-1 RNA levels showed that zygotic fem-1 activity in the germline was impaired in the absence of maternal fem-1 RNA.

The authors went on to show that the severity of the Fog phenotypes observed in heterozygotes depended on the history of the wild-type fem-1 allele. Repeatedly backcrossing heterozygotes with homozygotes increased the penetrance of Fog phenotypes in heterozygotes. This showed that although the wild-type allele remains the same genetically, it becomes heritably compromised epigenetically.

To explain these findings, the authors suggest that in the absence of maternal fem-1 RNA, the paternally contributed wild-type allele becomes epigenetically silenced in the zygotic germline. They perspicaciously reasoned that the anti-silencing activity of the maternal RNA may work by inactivating silencing siRNAs, hence licensing zygotic expression. The cluster of recent papers (discussed here) have come to similar conclusions, suggesting the presence of an anti-silencing pathway, possibly mediated by CSR-1 and it’s associated 22G-RNAs.

This epigenetic licensing system effectively marks genes previously expressed in the germline as ‘self’, whilst transcripts without a history of expression are silenced as potentially deleterious. So far the mechanisms of this pathway are yet to be elucidated. As the CSR-1/22G-RNA system targets thousands of genes, one would expect similar phenomena to those observed with fem-1 to be more regularly observed. I’m looking forward to further findings in this field, and potentially analogous systems being discovered in other organisms.

Johnson CL, & Spence AM (2011). Epigenetic licensing of germline gene expression by maternal RNA in C. elegans. Science (New York, N.Y.), 333 (6047), 1311-4 PMID: 21885785

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

A dual purpose RNA and Hox regulation

A new paper in Plos Genetics shows that a long non-coding RNA regulates the expression of a Hox gene in Drosophila in cis. This finding suggests an explanation for the co-linearity displayed by Hox genes between genomic arrangement and expression pattern.

The Ultrabithorax mutant.

Hox genes are master-regulators of positional identity along the anterior-posterior axis throughout bilaterian animals. Hox genes are found in genomic clusters in which their 3′-5′ organisation mirrors their expression pattern along the A-P axis. This correspondence between body axis and genomic organisation is termed co-linearity. An important feature of Hox gene genetics is the phenomenon of ‘posterior prevalence’. In any given segment the gene that has it’s most anterior boundary of expression in that segment will define segmental identity. Hence, if that gene is not expressed the segment will take on a more anterior identity. Perhaps the clearest example of this phenomenon is the Ultrabithorax mutant in Drosophila, in which segments that would have generated abdominal structures instead take on a thoracic fate, leading to flies with two sets of wings.

The Hox gene cluster is actually divided into two partial clusters in Drosophila; the Antennapedia complex (ANT-C) and the Bithorax complex (BX-C). BX-C consists of three Hox genes responsible for posterior patterning in Drosophila, Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) spread over ~300kb, and has become a paradigm for the understanding on genetic regulation. Many transcriptional enhancers, maintenance elements (sites for the binding of Polycomb-group and Trithorax-group chromatin modulating complexes), and encoded microRNAs responsible for regulating the expression of the BX-C genes have been discovered. However, a complete picture of BX-C regulation is still far away. It’s been known since the 1980’s that much of BX-C is transcribed, but the significance of this finding is just emerging. Gummalla et al. have used classical genetics to characterise the role of one such non-coding RNA in relation to the expression of abd-A in the embryonic CNS.

Figure showing the expression of ABD-A (red), and ABD-B (green) in the embryonic CNS. Note the gap in PS13, that isn’t filled by derepressed ABD-A in this mutant.

abd-A is expressed in the embryonic epidermis and CNS in parasegments (PS) 7-12 but is excluded from PS13. In line with ‘posterior prevalence’, this was considered to be due to Abd-B repressing abd-A expression. A mutation that removes Abd-B, shows expression of abd-A expression extending into PS13. However, this mutation also removed some of the sequence downstream of the transcription unit of Abd-B. In flies homozygous for more subtle mutations affecting Abd-B, abd-A expression only spreads into PS13 epidermis and not the CNS.  Therefore, some function located in the genomic region downstream of Abd-B (termed iab-8), was necessary for abd-A repression in the PS13 CNS. Gumalla et al. knew that a long non-coding RNA (iab-8 ncRNA) was predicted to initiate in this area, and therefore set out to characterise it’s function.

A map of the abdominal half of the bithorax complex. the iab-8 ncRNA is shown in blue (note exon structure). Abd-B, and abd-A are in black and the position of the miR-iab-8 is shown.

iab-8 ncRNA is transcribed from virtually the entire region between Abd-B and abd-A, spanning 92kb. Mutations that truncate iab-8 ncRNA near the Abd-B end cause a derepression of abd-A expression in the PS13 CNS, but mutations affecting the end nearest abd-A display only subtle derepression. The difference between these two classes of mutants, appears to be the position of a microRNA encoded by iab-8 ncRNA, miR-iab-8. This suggested that miR-iab-8 was responsible for the repression of abd-A in PS13 CNS. However, mutants with this miRNA deleted did not display the complete derepression phenotype, rather a very weak derepression of abd-A. This showed that there must be a second, partially redundant function of iab-8 ncRNA, apart from producing miR-iab-8.

To test whether a second miRNA or a small polypeptide encoded by iab-8 ncRNA was responsible for this second function, Gummalla et al. missexpressed iab-8 ncRNA from another locus in PS 8-13. This had no effect on ABD-A expression, suggesting that no other trans-acting factor is encoded by the ncRNA. They then performed some complicated genetic experiments that showed that iab-8 ncRNA acts to repress abd-A is cis. They generated flies that contained a deletion of miR-iab-8 on one chromosome, and a truncated copy of the iab-8 ncRNA on the other. These flies do not produce any of the miRNA, but still produce the ncRNA on one chromosome, and yet abd-A is derepressed in PS13 CNS. When flies are generated with one copy of the BX-C deleted, and a deletion of miR-iab-8 on the other chromosome, abd-A is not derepressed.

The iab-8 ncRNA therefore acts to repress abd-A expression in CNS of PS13 through two different mechanisms: a trans-acting miRNA, and through a cis-acting process of transcriptional interference. Although it is possible that this process of cis-repression could act by iab-8 ncRNA recruiting gene silencing machinery that would act by heterochromatin formation or DNA methylation, the authors suggest that it is more likely that iab-8 ncRNA acts by somehow interfering with the abd-A promoter. This leads them to suggest that if this method of gene regulation was widely used within Hox clusters it could explain the link between posterior prevalence and co-linearity. In this case expression of a more anterior gene is blocked in posterior segments by a more ‘posterior’ transcript. Similarly an upstream ncRNA acts to repress Ubx (Petruk et al.2006). This method of transcriptional interference by readthrough of more posterior genes or by upstream ncRNAs would fix the arrangement of Hox genes in an ancestral cluster, and hence the co-linearity that is observed today.

Gummalla, M., Maeda, R., Castro Alvarez, J., Gyurkovics, H., Singari, S., Edwards, K., Karch, F., & Bender, W. (2012). abd-A Regulation by the iab-8 Noncoding RNA PLoS Genetics, 8 (5) DOI: 10.1371/journal.pgen.1002720

A Ribosome Code?

The ribosome, a universally conserved molecular machine that catalyses protein synthesis, has generally been considered to act constitutively. That is to say, that ribosomes act to translate mRNAs in the same way across all cells and developmental stages. Regulatory control of translation is predominantly exerted by the action of translation initiation factors, which guide the association of the ribosome with target mRNAs. The eukaryotic ribosome is composed of 4 RNA molecules and 79 different ribosomal proteins (RPs). A paper published last year by Kondrashov et al. has shown one RP (RPL38) specifically regulates the expression of a subset of mRNAs during embryonic development in the mouse. Together with findings from human genetic diseases and from other organisms, this data is suggestive of a ‘ribosomal code’ regulating translation.

Kondrashov et al. set out to discover what gene was responsible for causing the morphological defects found in a spontaneous mouse mutant, tail-short (Ts). These mice display skeletal patterning defects, including homeotic transformations (ie. the conversion of a tissue’s identity to that of a different tissue; in this case changes between the segmental identities of vertebrae and ribs). They also display eye and craniofacial defects, short and kinky tails, and wavy neural tubes. These phenotypes are only found in heterozygous mice (Ts/+); homozygotes die at implantation stages. By positional cloning, Kondrashov et al. found that the gene responsible for Ts was Rpl38.

Ts/+ mice display skeletal defects and transformations along the entire length of the anterior-posterior body axis. The key regulators of morphological identity along the A-P axis are Hox genes. Hox genes encode homeodomain-containing transcription factors, and are found in four genomic clusters in vertebrates. Loss of function mutations in, or missexpression of, Hox genes generally leads to homeotic transformations (most shockingly seen in the Drosophila mutants antennapedia and ultrabithorax). Kondrashov et al. therefore examined the expression of Hox gene transcripts in Ts/+ mouse embryos. Surprisingly, they found no changes in the levels or expression domains of the Hox genes.

Schematic representation of the axial skeleton of WT and Ts/+ mice. Defects are explained by the effects of corresponding Hox gene mutants.

The researchers then asked whether changes in translational control of Hox genes were responsible for the Ts/+ phenotypes. Using various techniques they showed that there were no changes in global protein synthesis. However, by using quantitative PCR on mRNAs that were purified with active ribosomes, they identified a subset of Hox genes that were translationally deregulated in Ts/+ embryos (Hoxa4; a5; a9; a11; b3; b13; c8; d11).  These findings were confirmed by observing protein levels for HOXA5, A11, and B13 in the Ts/+ mouse embryos. The majority of the Ts/+ axial skeleton phenotypes could be accounting for by the known effects of loss of function mutations in the Hox genes that were translationally deregulated.

It therefore appears that RPL38 is exerting a specialised control on the translation of specific Hox genes. In further experiments Kondrashov et al. find that RPL38 is likely facilitating the formation of the 80S (complete) ribosomal complex on specific mRNAs (the ribosome is made up of two subunits, the 40S subunit associates with the 5’UTR of the target mRNA first and is then joined by the 60S subunit to make a translationally competent ribosome). An important question is whether RPL38 exerts it’s function as part of the ribosome, or whether it has extra-ribosomal roles as well? By separating ribosomal from ribosome-free cytosolic fractions, Kondrashov et al, found that RPL38 was only ever found in the ribosome.

Ribosomal proteins have generally been considered as ubiquitously expressed cellular ‘housekeeping’ proteins. However, when the researchers examined Rpl38 expression, they found that transcripts were enriched in specific tissues. For instance, embryonic tissues that give rise to facial structures, as well as the neural retina, showed high levels of Rpl38 expression, correlating with the craniofacial and eye defects in Ts/+ mice. Likewise, Rpl38 was strongly expressed in the somites and the neural tube, the embryonic tissues giving rise to the vertebrae and the spinal cord respectively. Kondrashov et al. went on to examine the expression of 72 different ribosomal proteins in 14 different tissue and cell types. They found a large amount of heterogeneity in RP expression, suggesting that many have specialised, tissue specific roles.

A few obvious outstanding questions for future studies should be noted; Does RPL38 bind cis-regulatory sequence or structure elements within target mRNAs? and what are they? Do trans-acting factors also play a role? Other developmental questions also stand out. Hox genes are not involved in eye development, and it also seems unlikely that the Hox genes implicated in the trunk segmental effects are also responsible for the craniofacial defects. What other RPL38 mRNA targets are responsible for these phenotypes?

These experiments have therefore shown that RPL38 has transcript-specific roles in the control of translation, and that many RPs display heterogeneous expression patterns rather than the previously assumed ubiquity. Together these findings suggest that RPs are imparting a new level of specificity in the control of gene expression. They fit into a broader array of observations that hint at the existence of a ‘ribosome code’ in which alterations in the composition of ribosomes leads to their translational specialisation towards subsets of mRNAs. Diamond-Blackfan Anaemia is a human genetic disease caused by mutations in a number of ribosomal proteins. Patients display limb defects, cleft palates, growth failures and cancer predisposition. Likewise knockdown of multiple distinct RPs in zebrafish leads to a wide range of developmental defects and a high incidence of cancer. A possible explanation for these types of finding, is that highly proliferating tissues may be more sensitive to differences in the rate of protein synthesis. Hence, indirect effects on cell proliferation and apoptosis may lead to the morphological abnormalities. However, Kondrashov et al. have shown in this study of Ts/+, overall protein synthesis is not affected, and the effects on a subset of developmental patterning genes are responsible for the bulk of the phenotypes.

Ribosomal RNAs and proteins are also targets for extensive chemical modifications such as phosphorylation and methylation, most of which are as yet uncharacterised. Interestingly, another human genetic disease, X-linked Dyskeratosis Congenita, is probably caused by failures of rRNA modifications. By analogy with the levels of complexity see with regard to modifications and combinations of chromatin-associated histones, a ‘ribosome code’ imparting translational specificity by heterogeneity of RPs and modifications has the potential to be a hugely important level of regulatory control.

Kondrashov, N., Pusic, A., Stumpf, C., Shimizu, K., Hsieh, A., Xue, S., Ishijima, J., Shiroishi, T., & Barna, M. (2011). Ribosome-Mediated Specificity in Hox mRNA Translation and Vertebrate Tissue Patterning Cell, 145 (3), 383-397 DOI: 10.1016/j.cell.2011.03.028

Topisirovic, I., & Sonenberg, N. (2011). Translational Control by the Eukaryotic Ribosome Cell, 145 (3), 333-334 DOI: 10.1016/j.cell.2011.04.006