To identify genes asymmetrically expressed between left and right sides in the developing embryo we compared gene expression using the MRC Mouse Known Gene Oligo Array printed array slides (Mm_SGC_Av2), which identify 7455 known loci. Left and right lateral plate tissue was dissected from 3-6 somite embryos and pools from 4 embryos were used to prepare RNA. Following SMART PCR amplification, hybridisation and analysis were performed as described in the Materials and Methods. The results from 4 replicates were analysed, and lists ranking the apparent degree of left or right sided asymmetry were generated (see Additional file 1). The presence, at the top of the left-sided list of the known left specific gene, Pitx2, demonstrated the validity of the approach. Significantly, Nodal, though present on the array, was not identified; Lefty2 was not present on the array. We further analysed expression of 7 loci from the left-sided and 6 from the right sided list by RNA wholemount in situ hybridisation (WISH) on 8.5 dpc embryos. Of the 13 loci examined, only one, Ablim1, showed apparent L-R asymmetry of expression (Fig. 1 and data not shown). Expression in the left LPM was clearly stronger and more extensive than in the right, while a second asymmetric domain was visible at the node. Initial analysis of a few embryos showed expression predominantly on the right hand side of the node.

Performing WISH using the full cDNA, we examined Ablim1 expression in a developmental series of embryos, from 6.5 dpc to 9.5 dpc (Fig. 1b-e). Expression was detected in the yolk sac of 6.5 dpc embryos, initially as patches (Fig. 1b) that became a distinct ring of expression by 7.5 dpc (Fig. 1c), consistent with it marking the prospective blood islands. At 7.5 dpc, expression was also seen in the developing head folds (Fig. 1c), an expression pattern maintained through 9.5 dpc (Fig. 1d, e). Ablim1 expression in the node was seen from 7.5 dpc (Fig. 1c). By 8.5 dpc L-R symmetric expression was evident in the developing heart. Strikingly, asymmetric expression was also seen in the LPM; stronger and more extensive on the left than the right (Fig. 1d). By 9.5 dpc expression was evident in the head, the somites and portions of the heart, but no L-R asymmetry was seen at this stage (Fig. 1e, data not shown).

Analysis of the published data 13 and ESTs http://www.ensembl.org demonstrates the existence of multiple Ablim1 transcripts, exhibiting both alternative splicing and multiple alternative first exons. Northern blot and RT-PCR analysis confirmed the existence of Ablim1 transcripts in 7.5 and 8.5 dpc embryos (data not shown). While multiple protein isoforms of Ablim1 exist, they comprise two major classes; long forms containing lim domains and a villin head piece (VHP) and a short form lacking the lim domains 13 (Fig. 1a). We investigated the temporospatial distribution of the transcripts encoding these isoforms using WISH probes hybridising to different portions of the Ablim1 message; probes corresponding to the beginning (Ex 2-6), the common region (Ex 8-25) and the 3'UTR (3'UTRa and b; Fig. 1). The Ex 2-6 probe, encompassing the lim domains, revealed expression in the developing heart (Fig. 1f) as well as the right side of the node when WISH colour development was extended for longer times (Fig. 1j), but not in the lateral plate. The Ex 8-25 probe revealed asymmetric expression to the right side of the node and in the left lateral plate; symmetric expression was evident in the head and heart (Fig. 1g, k). The 3'UTRa probe showed similar expression to Ex8-25, although node expression was significantly weaker when compared to the other expression domains (Fig. 1h, l). The terminal 3'UTR probe, 3'UTRb, showed similar expression, but failed to detect node expression, arguing for a shorter 3'UTR in the node transcripts (Fig. 1i, m). Together these results argue that a transcript encoding a long isoform containing both lim domains and VHP is present in the node, while a shorter isoform (with no lim domains) is present in the left lateral plate; it is not possible to say from our WISH or RT-PCR analysis whether the short isoform is also present in the node.

Nodal, often thought of as the master gene controlling left-sided gene expression, is expressed in the left but not right lateral plate from 3-6 somite stages 1415. If asymmetric Ablim1 expression is directly controlled by Nodal, it would be expected to exhibit similar temporospatial expression and not be expressed asymmetrically prior to asymmetric Nodal expression. We therefore examined the temporospatial expression of Ablim1 from the late headfold to the 10 somite stage by WISH (using the 3'UTRa probe). Up to and including the 2 somite stage, bilaterally symmetrical expression was seen in the anterior lateral plate, contiguous with expression in the heart (Fig. 2a). By 3 somites expression in the LPM became clearly asymmetric, with expression in the left lateral plate being both stronger and stretching noticeably further posteriorly than in the right (Fig. 2b). This asymmetric expression was highly evident at 5 somites, being particularly obvious when the WISH is developed for a short time (Fig. 2c). By 7 somites, after lateral plate Nodal expression has ceased, asymmetric left lateral plate expression of Ablim1 was strongly downregulated, although still evident at a low level in some embryos (Fig. 2d). By 8 somites, no sign of asymmetric lateral plate expression was evident (Fig. 2e). Analysis of sections revealed that lateral plate expression was present throughout the lateral plate mesoderm (Fig. 2f, g) similar to Nodal. These data are consistent with the hypothesis that Nodal activates Ablim1. As with our previous results, expression of Ablim1 in the node was apparent when the WISH colour development was left for longer periods of time (Fig. 1h, l, 2b).

To further test the hypothesis that Nodal activates asymmetric Ablim1 expression, Ablim1 lateral plate expression was analysed in mutants with abnormal L-R patterning. Dnahc11 encodes a dynein heavy chain required for nodal cilia motility: the point mutant Dnahc11^iv (iv) results in immotile nodal cilia, absence of nodal flow, and randomisation of both situs and Nodal lateral plate expression 15161718. When Ablim1 lateral plate expression was analysed in iv mutants, a mixture of expression patterns were seen (Table 1), similar to those previously reported for Nodal in the iv mutant 15, including left-sided (Fig. 3a), bilateral (Fig. 3b) and right sided (Fig. 3c) expression. These data show that Ablim1 asymmetry is downstream of nodal flow, similar to Nodal asymmetry and is consistent with Ablim1 asymmetry being downstream of Nodal. This hypothesis was further supported by analysis of Shh mutant embryos; Shh mutants do not express the Nodal antagonist Lefty1 in the midline, resulting in bilateral Nodal expression 19. Consistent with a role for Nodal upstream of lateral plate Ablim1 expression, bilateral Ablim1 expression was seen in Shh^-/- embryos (data not shown and Table 1).

In the lateral plate, Nodal's auto-activation of its own expression, as well as its activation of Lefty2 and Pitx2 expression, is mediated by binding of FoxH1 to asymmetric elements (ASEs). ASEs contain two or three FoxH1 binding sites (TGT G/T T/G ATT) within a 30-200 bp region. The random frequency of a pair of binding sites within such a region is once every 350 kb. When ASE-like sequences were sought around the mouse and human Ablim1 loci, 7 sequences were identified in mouse and 3 in humans (see Additional file 2), although position and overall sequence was not conserved. To address whether Nodal might be interacting with Ablim1 through these elements, the sequences plus 100 bp on either side were PCR amplified and cloned into luciferase reporter vectors. These were transfected into HepG2 cells together with FoxH1 and a constitutively active Alk4 construct. While a control fragment from the mouse Pitx2 ASE activated luciferase, as previously reported 24, all the Ablim1 derived fragments failed to activate expression above background levels, arguing that Nodal does not activate Ablim1 through FoxH1 binding to an ASE (data not shown).

When temporospatial Ablim1 expression was analysed in the node, utilising the Ex2-6 probe, transcript was detected from when the node is first patent (Fig. 4a, b). Intriguingly, this first expression is of a salt and pepper pattern stretching across the pit of the node. This resolved to give a ring surrounding the node by the mid-headfold stage (Fig. 4c); the first indication of asymmetry was evident at this stage, with a higher level of expression on the right than the left hand side of the node (Fig. 4c). During the next few hours of development, expression on the anterior left side of the node was lost, while expression was activated in the midline cells anterior to the node, resulting in a question mark-like expression pattern (Fig. 4d). By 4 somites, the remaining left-sided expression was lost, resulting in solely right-sided expression at the node (Fig. 4e). This abrogation of expression continued in a clockwise direction around the node (Fig. 4f), until by 7 somites peri-nodal expression was restricted to midline cells anterior to the node (Fig. 4g). By 8 somites midline expression has also been lost (Fig. 4h). Sections through these embryos reveal that expression in the node is restricted to the ventral layer (Fig. 4i).

The early dynamic expression pattern of Ablim1 in the node correlates with the changes in nodal cilia motility and nodal flow described by Okada 18. The loss of Ablim1 expression from the pit corresponds to the stage when local vortices form, while the loss of expression from the left side of the node correlates with the establishment of a strong leftwards flow. In conjunction with the very early asymmetry of Ablim1, this suggests that expression may be responding directly to nodal flow. To test this hypothesis we next examined Ablim1 node expression in iv mutants, where there is no nodal flow. Of 40 mutant embryos analysed, no asymmetry was detected at any stage of development. Indeed, the robust peri-nodal ring of expression was never detected. There was, however, a rise in the number of embryos where expression was not detected, from 12% in wt to 42% in iv/iv mutants (Table 3). Intriguingly, in the 58% of embryos where expression was detected, the pattern of expression was the same patchy expression seen in the very earliest wild type nodes (Fig. 5a). This pattern was maintained well through the period that leftwards laminar flow is normally detected and that strong asymmetry of Ablim1 is normally seen at the node. These data demonstrate that nodal flow is required for the upregulation of Ablim1 expression in the peri-nodal region and is involved in downregulation of Ablim1 expression in the pit of the node.

Work from many research groups over the past decade has established a generally accepted pathway underlying establishment of L-R patterning in mammals (reviewed 4). The activation of the Nodal signalling cascade in the left LPM results in asymmetric, left-sided, expression of Pitx2 that is maintained into organogenesis (Fig. 6). In light of misexpression experiments in chick and Xenopus, Pitx2 has been argued to specify left sidedness 1112. Yet, while Pitx2 mutant mouse embryos demonstrate right pulmonary and atrial isomerism 25262728, the initial direction of embryonic turning and heart looping are Pitx2-independent 22. In contrast, analysis of mice lacking or unable to respond to Nodal signalling in the lateral plate showed a randomised direction of heart looping 2329; the direction of embryonic turning was also randomised in Cryptic (MGI: Cfc1) mutants, but not reported for the Nodal mutants 2329. These data argue that heart looping and embryonic turning are controlled by Nodal expression, however, it is not possible to distinguish the role of Nodal at the node from its role in the lateral plate in these experiments. Our results clearly show Ablim1 asymmetry is independent of Pitx2 expression. More significantly, Ablim1 is capable of being asymmetrically expressed in the absence of detectable LPM Nodal signalling. Quite clearly, another L-R asymmetric signal is present in the embryo, that for the sake of discussion we shall refer to as "signal X".

While it is clear from our analysis that Ablim1 can be asymmetrically expressed in the absence of detectable Nodal and Pitx2, asymmetric Nodal obviously does play a role. The strong reduction of lateral plate Nodal expression in Nodal^D600/D600 embryos results in ~50% of embryos with detectable asymmetric LPM Ablim1 expression, while the total removal of LPM Nodal expression in the Nodal^Δnode/- embryos reduces this level to 24% (Table 1). The level of LPM Nodal expression therefore directly affects asymmetric Ablim1 expression. This relationship is underlined when the temporal activation of Ablim1 is analysed; strong reduction of Nodal signalling is known to result in delayed target gene activation 20. At early somite stages no Nodal-independent Ablim1 expression is seen, compared to expression in 30% of embryos by 4-5 somites and 50% by 6-7 somites (Table 2). It is therefore clear that two signals, a Nodal-dependent and the Nodal-independent "signal X" influence asymmetric lateral plate Ablim1 (Fig. 6A). While we have drawn "signal X" and Nodal both directly acting on Ablim1, we cannot rule out the possibility that "signal X" is in part regulated by Nodal. It is also possible that there is a temporal offset between Nodal signalling and "signal X", with Nodal acting first. The data neither argue for nor against these scenarios and in the absence of identifying and interfering with "signal X" it is impossible to distinguish between them.

A signal acting on the left side of the embryo may reflect either a left-sided activator or a right sided repressor of Ablim1 expression. Work from various groups has revealed additional lateral plate asymmetries in gene expression. BMP signalling, as assessed by Smad1 phosphorylation, is L-R asymmetric and this asymmetry is driven by asymmetric expression of the BMP antagonists chordin and noggin, which in turn are controlled by Nodal 30. Therefore, it seems unlikely that BMP signalling lies upstream of Ablim1 asymmetry. Similarly, the Nkx3-2 (or BapX1) homeodomain locus shows right sided asymmetric expression, but again is thought to act downstream of Nodal 31. In contrast, little is known about control of the right sided transcription factor Snail (Snai1), which temporally mirrors Nodal 32. Conditional deletion leads to randomised embryonic turning and heart looping and bilateral activation of the Nodal signalling cascade, arguing that it normally inhibits right sided Nodal expression. It is therefore possible that Snai1 inhibits Ablim1 expression on the right side of the embryo. Indeed the role of the snail family of genes as transcriptional repressors is well documented 33. A formal possibility exists that "signal X" in fact represents a very low level Nodal signal, below that capable of activating the known LPM targets of Nodal. While no Nodal expression is detected in the LPM, low level ectopic Nodal expression was reported in a few cells in the pit of the node in a small proportion of Nodal^Δnode/- embryos 23. Such a localised signal would be expected to first activate expression close to the node, in a manner similar to that reported for initial Nodal activation [discussed in 4. No such localised expression was evident for Ablim1 in Nodal^Δnode/- embryos (Fig. 3g and data not shown). Indeed, such putative low level Nodal signalling falls outside of the known activity of Nodal and it too would arguably constitue a novel asymmetric signal.

Wild type Ablim1 expression in the node changes markedly at the mid-late headfold stage, from a low level broad pan-node expression to a robust peri-nodal ring (Fig. 4). This reflects two changes; upregulation in the crown cells surrounding the node and downregulation within the pit of the node. Developmentally this corresponds to when nodal cilia are driving vortical fluid motion 18, suggesting that cilia driven fluid flow plays a role in these expression changes. This is supported by the failure of iv and Pkd2 mutant embryos to express the peri-nodal ring of Ablim1 expression (Table 3). Moreover, almost 60% of these embryos maintain the same low level pan-node expression seen in early wild type embryos. We detected no expression in nodes of the remaining mutant embryos and argue that this reflects either failure to maintain low level pan-node expression over time and/or technical limitations in detecting low level gene expression by WISH. The role of nodal cilia motility in generating nodal flow and L-R asymmetry is so central to thinking that little consideration has been given to any earlier role for fluid flow. Our data, however, reveals an early and previously unrecognised function for nodal flow in modulating symmetrical gene expression within the node, separate from the role of nodal flow in L-R determination.

The first L-R asymmetry in Ablim1 expression is evident in the node by the late headfold stage (Fig. 4c), several hours before the 1-2 somite stage at which asymmetry is evident for the other known asymmetric loci Nodal, Cerl2 (MGI: Dand5) and Lplunc1 1415343536. This argues that Ablim1 is responding to very early asymmetric signals. Nodal flow is argued to be the initial asymmetric signal in the mouse (reviewed 4) and is clearly driving fluid flow leftwards by early somite stages 18. However, the first Ablim1 asymmetry is evident at a stage when beads introduced into a node in vitro are carried leftwards only inefficiently and on average, hopping from vortex to vortex 18. Whether such an inefficient flow could affect sensory cilia sufficiently to fulfil the requirements of the two cilia hypothesis is unclear. Presumably NVPs could be carried leftwards in a similar manner to the beads, although whether they would break efficiently in such a flow is uncertain.

By the early somite stages a completely novel and very distinctive asymmetry of Ablim1 expression becomes evident at the node. The peri-nodal ring "retreats" around the node in a clockwise direction between 3 and 7 somites. This asymmetry of expression is very different from that seen for other asymmetrically expressed loci at the node; these show bi-lateral expression flanking the node, with stronger expression on one side than the other.

While the control of Ablim1 node asymmetry is not addressed by this study, the speed of the changes in Ablim1 expression suggests that this is an active process. In chick an anti-clockwise migration of cells around the node underlies gene asymmetry 3738. However, in mice, cre-loxP based lineage analyses of both node crown and pit cells did not reveal such cell migration 2339. The role of flow in controlling earlier changes in Ablim1 expression makes flow a mechanism we must contemplate. Yet for flow to control Ablim1 asymmetry, the following objections must be taken into account. (1) How could leftwards flow initially affect just the anterior node? At early somite stages the anterior node is shallower than the posterior 4041 and this may influence the ability of flow to impact on the crown cells in the anterior versus the posterior. (2) How does flow subsequently affect the posterior node? As the embryo grows the node remodels, becoming more even in depth between anterior and posterior. At the same time leftwards flow becomes laminar. A combination of these two events may then allow flow to also affect posterior left-sided node crown cells. (3) How does flow subsequently affect the right side of the node? In vivo, fluid flow within the node recycles being drawn downwards on the right hand side 42. A combination of growth, node remodelling and perhaps temporal accumulation of signalling may allow the right side of the node to respond to the recycled flow as it is pulled back into the right hand side of the node (Fig. 6B).

While there is strong evidence for nodal flow in many vertebrates 434445, earlier, pre-flow events have been demonstrated to influence L-R patterning in non-mammalian species. Vg1 can influence situs determination and its putative co-receptor, Syndecan-2, becomes asymmetrically phosphorylated in pre-flow Xenopus embryos 46474849. Pharmacolgical experiments have implictaed H⁺K⁺ATPase, VATPase, Serotonin and 14-3-3 family member E in Xenopus situs determination 505152. In the resulting serotonin model, an electric field drives serotonin through gap junctions in Xenopus, resulting in higher right than left sided localisation 525354. The H⁺K⁺ATPase mRNA similarly becomes asymmetrically localised in Xenopus and perturbed expression disrupts L-R patterning in both Xenopus and chick 52. Therefore the question must be raised as to whether such early mechanisms also exist in mammals and may be controlling Ablim1 asymmetry, either at the node or in the LPM.

That there is asymmetry of Ablim1 expression at both the node and the LPM bears comparison to the expression pattern of Nodal. Nodal asymmetry at the node slightly predates that in the LPM and is required for LPM expression in the mouse 23. It has even been argued, in light of the ability of Nodal to autoactivate, that Nodal at the node might be carried to left LPM to activate expression there 55. In striking contrast, Ablim1 asymmetry is on opposite sides in the node and LPM. So while there is Ablim1 asymmetry at the node when LPM asymmetry is first detected, the expression domains are on opposite sides of the embryo (Fig. 1g). Moreover, while Nodal is a signalling molecule, Ablim1 is a structural, cell autonomously acting protein. It is difficult to envisage how a cytoskeletal protein would be acting to repress its own expression across many cell diameters. More likely, the two expression domains are independently regulated. Indeed, the long isoform of the protein seen at the node but not detected in the LPM originates at an alternate first exon, consistent with different promoter enhancer combinations controlling the two expression domains.

Uniquely, for mammalian asymmetric genes, Ablim1 encodes a structural protein. As its name suggests, Ablim1 protein binds to actin and when first identified, the presence of lim domains led the authors to suggest that it might act as an adaptor protein, bringing other proteins to the actin cytoskeleton 56. The homologue in C. elegans, unc-115, similarly binds actin 57, and when mutated leads to an uncoordinated phenotype and defects in axon guidance 58. Expression of a dominant negative Ablim1 in chick embryos leads to similar axonal phenotypes 59. Yang and Lundquist 60 further demonstrated that expression of unc-115 in mammalian fibroblasts led to the formation of peripheral actin conglomerations at the expense of stress fibres. One possible explanation that they suggest is that Ablim1 protein may have different roles at the cell membrane and in the cytoplasm, acting to abrogate stress fibre formation when cytoplasmic. This raises the possibility that asymmetric Ablim1 expression might prove permissive for asymmetric morphogenetic changes in the embryo. However, when an isoform specific deletion of Ablim1 was made, for the isoform seen in the eye, no defects were reported 13. This deletion, however, seems unlikely to affect the expression that we have described. Indeed many additional alternative first exons are now annotated that were not evident to Lu and colleagues.

To collect staged embryos, mating was assessed by monitoring for copulation plugs. The day of plug was designated day 0.5. Wild type embryos were (C3H/HeH × 101/H)F1s. Mutant lines were: iv- Dnahc11^iv 6162; Pkd2^lrm4 63; Nodal^D600, Nodal^tm2Rob 20; Nodal^- is the Nodal-LacZ allele Nodal^tm1Rob 14; Nodal^Δnode is the Nodal node del, Nodal^tm3Rob 23; Pitx2c null, Pitx2^tm3.1Jfm 22; Shh, Shh^tm1Chg 64.

All animals were used in accordance with UK Home Office regulations.

Tissue for micro-array analysis was dissected in cooled PBS, then snap frozen in liquid nitrogen. RNA was produced by Qiagen RNeasy mini kit and quality assessed by Agilent 2100 Bioanalyzer. Reverse transcription and amplification were conducted according to the SMART mRNA Amplification Kit (Clontech). The resulting samples were labelled with Cy3 and Cy5 and used to hybridise MRC Mouse Known Gene Oligo Array printed array slides (Mm_SGC_Av2), identifying 7455 known genes, according to standard protocols. Results from 4 experimental repeats were analysed using GeneSpring (Agilent Technologies). Genes were ranked for differences in left versus right sided expression. The data from these experiments has been submitted to ArrayExpress, reference E-MEXP-2277.

The following IMAGE clones (IC) were used to produce anti-sense WISH probes: IC1-3995027 Oaz1, IC2-4457726 Agtrl1, IC3-4935411 Ablim1, IC4-5042041 Tssc3, IC5-5123607 F10, IC6-5291579 Fkbpla, IC7-5329923 Cts1, IC8-5716506 Plk, IC9-6335891 Hmgi, IC10-6390454 Aqp3, IC11-3963483 Tpm1, IC12-5715646 tuba1, IC13-5685601 Hyal1.

IC3 represents a full length Ablim1 clone. Clones portions of Ablim1 sequence, exons 2-6, 8-25 and 3'UTR sequences, were produced by restriction digest or PCR amplification and cloned into pBluescript2. Antisense RNA probes were produced and used for wholemount in situ hybridisation, according to standard protocols.

Putative ASE sequences were amplified by PCR from BALB/c mouse and commercial human DNA, TA cloned into pGL3-Promoter vector (Promega). The sequence cloned into pGL3 was confirmed by sequencing. Luciferase assays were carried out as previously described 65.