Bilateral Right Sidedness Sequence


About Right Sidedness Sequence

Right atrial isomerism is a severe complex congenital heart defect resulting from embryonic disruption of proper left-right axis determination. RAI is usually characterized by complete atrioventricular septal defect with a common atrium and univentricular AV connection, total anomalous pulmonary drainage, and transposition or malposition of the great arteries.

Affected individuals present at birth with severe cardiac failure. Other associated abnormalities include bilateral trilobed lungs, midline liver, and asplenia, as well as situs inversus affecting other organs. Left atrial isomerism (LAI) is a related disorder with a somewhat better prognosis.

LAI is characterized by bilateral superior vena cava, interruption of the intrahepatic portion of the inferior vena cava, partial anomalous pulmonary venous drainage, and ventricular septal defect.

Patients with LAI may have polysplenia and bilateral bilobed lungs, as well as situs inversus affecting other organs. Both RAI and LAI malformation complexes have classically been referred to as Ivemark syndrome (summary by Eronen et al., 2004 and Kaasinen et al., 2010).

With the z-transform, the s-plane represents a set of signals (complex exponentials). For any given LTI system, some of these signals may cause the output of the system to converge, while others cause the output to diverge (“blow up”).

The set of signals that cause the system’s output to converge lie in the region of convergence (ROC). This module will discuss how to find this region of convergence for any discrete-time, LTI system.

Symptoms of Bilateral right-sidedness sequence

Clinical features: Imported from Human Phenotype Ontology (HPO)

  • Abnormality of the abdomen
  • Asplenia
  • Polysplenia
  • Abnormality of the cardiovascular system
  • Atrial septal defect
  • Common atrium
  • Pulmonary artery atresia
  • Pulmonary venous return anomaly
  • Right atrial isomerism
  • Situs inversus totalis
  • Ventricular septal defect


Role of ASE in the asymmetric expression of nodal

Previously, we have shown that L-R asymmetric expression oflefty-2 is attributable to ASE, whereas that oflefty-1 is mediated by a combination of bilateral enhancers and a right side-specific silencer (Saijoh et al. 1999). We have now shown that expression of nodal, like that of lefty-2, is regulated by a left side-specific enhancer (Fig.). Similar results have been obtained by others (Norris and Robertson 1999).

It is not absolutely clear whether ASE alone is responsible for the asymmetric expression of nodal. Although targeted deletion of ASE in embryonic stem cells largely prevented the asymmetric expression of nodal, weak left-sided expression was still apparent in the left LPM (Norris and Robertson 1999).

Furthermore, in our transient transgenic assay, 5′-1, a construct lacking ASE, repeatedly gave rise to faint X-gal staining in a few cells in the left LPM. The permanent line N1, harboring the 5′-2 transgene, also showed similar weak lacZ expression in the left LPM.

Thus, although ASE appears to have the major role, an additional enhancer (LSE, for left side-specific enhancer) in the proximal promoter region (between −5 and 0 kb) likely also contributes to the asymmetric expression of nodal. Although ASE and LSE might each act independently as a left side-specific enhancer in our transgenic assays, they may function synergistically to establish asymmetric expression of nodal in vivo.

The expression of nodal in the PFP has not been described previously. However, the nodal ASE was able to direct left-sided expression of lacZ in the PFP. This effect was most apparent when the nodal ASE was linked to the hsp68promoter.

For several reasons, we believe that the ASE-induced expression in the PFP is not an artifact of the transgenic assay, but that it rather reflects expression of nodal in the PFP in vivo: (1) X-gal staining in the PFP was repeatedly observed with3′-1hsp and its deletion mutants in transient as well as permanent transgenic embryos; (2) X-gal staining in the PFP showed an apparent L-R specificity, being observed predominantly in a few cells on the left side, similar to the expression patterns oflefty-1 and lefty-2; and (3) X-gal staining in the PFP, like that in LPM, responded to iv, inv, andlefty-1 mutations.

We attempted to detect nodal mRNA in the PFP by whole-mount in situ hybridization; however, although weak signals were detected occasionally in the PFP, we were unable to obtain convincing results (data not shown). It is likely that the extent ofnodal expression in the PFP is not sufficient to be detected by this method.

A similar reason may also explain why X-gal staining in the PFP was not obvious with 3′-1 (the hsp68promoter appeared more efficient than did the nodal promoter, given that the intensity of X-gal staining in the left LPM induced by3′-1hsp was greater than that induced by3′-1).

An alternative possibility is that although ASE possesses an intrinsic ability to induce expression in the PFP,nodal expression is normally repressed in PFP by a negative regulatory element. nodal expression in the PFP may be negatively regulated by Lefty-1 because 3′-1 transgene gave rise to X-gal staining in the absence of lefty-1.

The 3.5-kb intronic fragment containing ASE responded to iv,inv, and lefty-1 mutations. Expression of the3′-1hsp (and 3′-1) transgene was affected in a manner similar to that of nodal itself, as determined previously by in situ hybridization (Collignon et al. 1996; Lowe et al. 1996; Meno et al. 1998). It is therefore likely that signals derived from iv, inv, and lefty-1 converge within the ASE region of nodal.

Our data suggest that the expression of nodal in the node is regulated by a separate enhancer (NDE) located in the far-upstream region. Although nodal expression in the node exhibits subtle L-R specificity (Collignon et al. 1996), constructs 5′-2and SN5.0, as well as deletion mutants derived fromSN5.0, failed to show L-R specificity of expression in the node.

Thus, NDE itself does not appear to possess L-R specificity. Such asymmetric expression of nodal in the node may be achieved by a combination of NDE and an additional cis-regulatory element such as ASE.

Similarities between nodal ASE and lefty-2 ASE

The ASEs of lefty-2 and nodal exhibit several common features: (1) Both enhancers direct left-sided expression not only in the LPM and but also in the PFP; (2) they both respond toiv, inv, and lefty-1 mutations (Fig. ;Saijoh et al. 1999); and (3) with both enhancers, directed expression in the PFP can be partially separated from that in the LPM.

One difference between the two enhancers is that whereas thelefty-2 ASE (350-bp region) can be clearly divided into at least two subdomains (one for the anterior LPM, the other for the posterior LPM), no such subdivision was apparent for nodal ASE (340 bp region).

Finally, several conserved sequence motifs are apparent in the nucleotide sequences of the two enhancers. Thus, the asymmetric expression of nodal and of lefty-2 may be regulated by similar transcriptional mechanisms.

Dissection of the 340-bp region of the nodal ASE suggested that most (if not all) of the conserved motifs are required for maximal enhancer activity. However, a 7-bp conserved sequence (CCCTGCC) located in the 3′ portion of this region may be particularly important for expression in the PFP. Moreover, a 10-bp conserved sequence (CAATCCACAT), which exist twice in nodal ASE andlefty-2 ASE, appears essential for expression in both left LPM and PFP.

However, database searches (TRANSFAC) failed to identify any previously known transcription factor binding sites that corresponded to these sequences. Clarification of the mechanism by which signals derived from upstream genes, such as the inv and ivgenes, induce the asymmetric expression of nodal will require identification of the transcription factors that bind to these conserved sequences.


We thank Dominic Norris and Elizabeth Robertson for communicating their unpublished data prior to submission; Chikara Meno forlefty-1 mutant mice; Kazuko Miyama, Tomoe Tanabe, and Yasuko Kasakawa for excellent technical and secretarial assistance; Atsusi Kuroiwa for mouse nodal cDNA; Alexandra Joyner and Janet Rossant for hsp68–lacZpA; Takahiko Yokoyama and Paul Overbeek for inv +/− mice; and Martina Brueckner for information on iv genotyping. This work was supported by CREST JST, and by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked ‘advertisement’ in accordance with 18 USC section 1734 solely to indicate this fact.

In our transgenic assays, ASE appeared to be both essential and sufficient for the asymmetric expression of nodal. In general, transgenic or transfection assays often overestimate the role of acis-regulatory element.

However, targeted deletion of a 600-bp region spanning the nodal ASE almost abolished the left-sided expression of nodal, confirming the critical role of ASE in the asymmetric expression of this gene (Norris and Robertson 1999).

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