Role In Left-right Patterning Research Articles

Molecular Pathways and Animal Models of Defects in Situs.

Left-right patterning is among the least well understood of the three axes defining the body plan, and yet it is no less important, with left-right patterning defects causing structural birth defects with high morbidity and mortality, such as complex congenital heart disease, biliary atresia, or intestinal malrotation. The cell signaling pathways governing left-right asymmetry are highly conserved and involve multiple components of the TGF-β superfamily of cell signaling molecules. Central to left-right patterning is the differential activation of Nodal on the left, and BMP signaling on the right. In addition, a plethora of other cell signaling pathways including Shh, FGF, and Notch also contribute to the regulation of left-right patterning. In vertebrate embryos such as the mouse, frog, or zebrafish, the specification of left-right identity requires the left-right organizer (LRO) containing cells with motile and primary cilia that mediate the left-sided propagation of Nodal signaling, followed by left-sided activation of Lefty and then Pitx2, a transcription factor that specifies visceral organ asymmetry. While this overall scheme is well conserved, there are striking species differences, including the finding that motile cilia do not play a role in left-right patterning in some vertebrates. Surprisingly, the direction of heart looping, one of the first signs of organ left-right asymmetry, was recently shown to be specified by intrinsic cell chirality, not Nodal signaling, possibly a reflection of the early origin of Nodal signaling in radially symmetric organisms. How this intrinsic chirality interacts with downstream molecular pathways regulating visceral organ asymmetry will need to be further investigated to elucidate how disturbance in left-right patterning may contribute to complex CHD.

Mink1 regulates spemann organizer cell fate in the xenopus gastrula via Hmga2

Congenital Heart Disease (CHD) is the most common birth defect and leading cause of infant mortality, yet molecular mechanisms explaining CHD remain mostly unknown. Sequencing studies are identifying CHD candidate genes at a brisk rate including MINK1, a serine/threonine kinase. However, a plausible molecular mechanism connecting CHD and MINK1 is unknown. Here, we reveal that mink1 is required for proper heart development due to its role in left-right patterning. Mink1 regulates canonical Wnt signaling to define the cell fates of the Spemann Organizer and the Left-Right Organizer, a ciliated structure that breaks bilateral symmetry in the vertebrate embryo. To identify Mink1 targets, we applied an unbiased proteomics approach and identified the high mobility group architectural transcription factor, Hmga2. We report that Hmga2 is necessary and sufficient for regulating Spemann's Organizer. Indeed, we demonstrate that Hmga2 can induce Spemann Organizer cell fates even when β-catenin, a critical effector of the Wnt signaling pathway, is depleted. In summary, we discover a transcription factor, Hmga2, downstream of Mink1 that is critical for the regulation of Spemann's Organizer, as well as the LRO, defining a plausible mechanism for CHD.

Biallelic DAW1 variants cause a motile ciliopathy characterized by laterality defects and subtle ciliary beating abnormalities

PurposeThe clinical spectrum of motile ciliopathies includes laterality defects, hydrocephalus, and infertility as well as primary ciliary dyskinesia when impaired mucociliary clearance results in otosinopulmonary disease. Importantly, approximately 30% of patients with primary ciliary dyskinesia lack a genetic diagnosis. MethodsClinical, genomic, biochemical, and functional studies were performed alongside in vivo modeling of DAW1 variants. ResultsIn this study, we identified biallelic DAW1 variants associated with laterality defects and respiratory symptoms compatible with motile cilia dysfunction. In early mouse embryos, we showed that Daw1 expression is limited to distal, motile ciliated cells of the node, consistent with a role in left-right patterning. daw1 mutant zebrafish exhibited reduced cilia motility and left-right patterning defects, including cardiac looping abnormalities. Importantly, these defects were rescued by wild-type, but not mutant daw1, gene expression. In addition, pathogenic DAW1 missense variants displayed reduced protein stability, whereas DAW1 loss-of-function was associated with distal type 2 outer dynein arm assembly defects involving axonemal respiratory cilia proteins, explaining the reduced cilia-induced fluid flow in particle tracking velocimetry experiments. ConclusionOur data define biallelic DAW1 variants as a cause of human motile ciliopathy and determine that the disease mechanism involves motile cilia dysfunction, explaining the ciliary beating defects observed in affected individuals.

MiR-430a regulates the development of left-right asymmetry by targeting sqt in the teleost.

microRNAs (miRNAs) are short, endogenous non-coding RNAs that contain approximately 18-22 nucleotides. miRNAs are involved in gene regulation by recognizing and binding the 3'UTR of target gene. In our previous data, miR-430 family showed significant differential expression modes through metamorphosis in Japanese flounder. It was speculated that miR-430a plays a key role in left-right patterning. We predicted the targets of miR-430a and gene ontology (GO) was performed. We speculated miR-430a is involved in the basal molecular function and organ development. In Japanese flounder, sqt as a target of miR-430a was enriched into heart development term. Sqt has been reported to participate in mesendoderm formation and organ development. Cardiac morphogenesis is the first asymmetric development process, which breaks left-right symmetry in bilateria. It was used as a marker to detect L-R asymmetric effects of miR-430a. Overexpression and suppression of miR-430a resulted in abnormal KV (Kupffer's vesicles) development and disordered in nodal-related expression with consequent cardiac laterality. Squint mRNA of Japanese flounder (Posqt) as a target of miR-430a was overexpressed and caused similar phenotype with miR-430a suppression group, such as longer cilia in KV and high range of clmc2 and spaw ectopic expression. Moreover, rescue experiments were performed and suggested that cardiac and KV defections, induced by overexpressing miR-430a, could be rescued by injecting Posqt mRNA. These results suggested that miR-430a regulates the development of left-right asymmetry by targeting sqt in the teleost.

Alkylglycerol monooxygenase, a heterotaxy candidate gene, regulates left-right patterning via Wnt signaling

Congenital heart disease (CHD) is a major cause of morbidity in the pediatric population yet its genetic and molecular causes remain poorly defined. Previously, we identified AGMO as a candidate heterotaxy disease gene, a disorder of left-right (LR) patterning that can have a profound effect on cardiac function. AGMO is the only known alkylglycerol monooxygenase, an orphan tetrahydrobiopterin dependent enzyme that cleaves the ether linkage in alkylglycerols. However, whether AGMO plays a role in LR patterning was unexplored. Here we reveal that Agmo is required for correct development of the embryonic LR axis in Xenopus embryos recapitulating the patient’s heterotaxy phenotype. Mechanistically, we demonstrate that Agmo is a regulator of canonical Wnt signaling, required during gastrulation for normal formation of the left – right organizer. Mutational analysis demonstrates that this function is dependent on Agmo’s alkylglycerol monooxygenase activity. Together, our findings identify Agmo as a regulator of canonical Wnt signaling, demonstrate a role for Agmo in embryonic axis formation, and provide insight into the poorly understood developmental requirements for ether lipid cleavage.

WDR5 regulates left-right patterning via chromatin-dependent and -independent functions.

Congenital heart disease (CHD) is a major cause of infant mortality and morbidity, yet the genetic causes and mechanisms remain opaque. In a patient with CHD and heterotaxy, a disorder of left-right (LR) patterning, a de novo mutation was identified in the chromatin modifier gene WDR5 WDR5 acts as a scaffolding protein in the H3K4 methyltransferase complex, but a role in LR patterning is unknown. Here, we show that Wdr5 depletion leads to LR patterning defects in Xenopus via its role in ciliogenesis. Unexpectedly, we find a dual role for WDR5 in LR patterning. First, WDR5 is expressed in the nuclei of monociliated cells of the LR organizer (LRO) and regulates foxj1 expression. LR defects in wdr5 morphants can be partially rescued with the addition of foxj1 Second, WDR5 localizes to the bases of cilia. Using a mutant form of WDR5, we demonstrate that WDR5 also has an H3K4-independent role in LR patterning. Guided by the patient phenotype, we identify multiple roles for WDR5 in LR patterning, providing plausible mechanisms for its role in ciliopathies like heterotaxy and CHD.

245 SUPERNUMERARY OUTFLOW TRACT CUSHIONS IN A MODEL OF TRANSPOSITION OF THE GREAT ARTERIES

Introduction Congenital heart defects are a leading cause of morbidity and mortality in new-borns; the most severe of which are abnormalities in the development of the outflow tract (OFT), such as transposition of the great arteries (TGA) and double outlet right ventricle (DORV). It has been previously suggested that TGA is caused by failed rotation of the OFT and/or abnormal spiralling of the two cushions that septate the OFT. Our aim was to investigate these possibilities using a murine model of TGA - the Pitx2c null ( Pitx2c −/− ) mutant. Pitx2 is a transcription factor with a key role in left-right patterning and cardiac development. Homozygous knock-out of the c-isoform in mice causes predominantly TGA, but also DORV, and is lethal at birth. Heterozygotes are viable, fertile and have no gross structural abnormalities. Method The Pitx2c mutant allele was generated as previously described by Liu et al. (2002). Pitx2c +/− and Pitx2c −/− embryos were collected at embryological ages E10.5, 11.5 and 12.5 days. The cardiothoracic region of each embryo was dissected and imaged using high resolution episcopic microscopy (HREM), an imaging technique allowing precise imaging of serial 2D surfaces throughout tissues which can then be compiled and reconstructed to provide accurate 3D visualisation of the sample. A computerised tracking pad was used to manually select regions of interest (ROIs) within serial images - the OFT cushions and outflow channels. Using the 9Amira9 software these ROIs were assembled as 3D reconstructions of three replicate hearts of each genotype at each age; the morphologies of which were compared. Results Hearts in the Pitx2c +/− were normal, with OFT channels that spiralled around one another in a left to right orientation, viewed distally. As previously described, Pitx2c −/− channels were straight, parallel and positioned dorso-ventrally distally; and the right ventricle, cushions and channels were each somewhat smaller. Unexpectedly, the Pitx2c −/− hearts also displayed novel endocardial cushion morphology. Not only were the cushions displaced in comparison to their Pitx2c +/− counter-parts, but two additional cushions were also found at 10.5 days. These cushions remodelled such that three remained at 11.5 days. At 12.5 days all Pitx2c −/− mice showed the typical morphology of the precursors of TGA or DORV. Conclusions We report for the first time the finding of supernumery OFT cushions in a model of TGA. These additional cushions were transient and appeared to resolve into two abnormally placed cushions which create non-spiralled outflow channels. There are other apparent abnormalities in the outflows of the Pitx2c −/− model including rotation and size reductions, so the exact role of the additional cushions in the development of TGA requires further study.

Zebrafish Nkd1 promotes Dvl degradation and is required for left–right patterning

The establishment of the left–right (LR) axis in zebrafish embryos relies on signals from the dorsal forerunner cells (DFC) and the Kupffer's vesicle (KV). While the Wnt signaling network influences many aspects of embryonic development, its precise role in LR patterning is still unclear. One branch of the Wnt network leads to stabilization of β-catenin and activation of downstream target genes. Other Wnt ligands appear to act independently of β-catenin to modulate calcium release and influence cell polarity. Central to regulation of β-catenin and coordination of convergent extension (CE) movements is Dishevelled (Dvl). Naked Cuticle (Nkd) binds Dvl and modulates β-catenin-dependent and independent Wnt signaling. Here, we analyze the expression patterns of three zebrafish Nkd homologs and find enriched expression of nkd1 in DFCs and KV. Dvl is degraded upon Nkd1 overexpression in zebrafish. Knockdown of Nkd1 specifically in the DFC results in β-catenin nuclear localization and transcriptional activation as well as alterations to DFC migration, KV formation, ciliogenesis and LR patterning. Furthermore, we identify asymmetric expression of the Nodal antagonist charon around the KV and show that Nkd1 knockdown impacts asymmetric charon expression. Our findings show that Nkd1 acts as a β-catenin antagonist in the DFCs necessary for LR patterning.

03-P001 Identification of an interaction domain in the Pitx2c N-terminus that is critical for its role in left-right patterning

03-P001 Identification of an interaction domain in the Pitx2c N-terminus that is critical for its role in left-right patterning

Cerberus is a feedback inhibitor of Nodal asymmetric signaling in the chick embryo

The TGF-beta-related molecule Nodal plays an essential and conserved role in left-right patterning of the vertebrate embryo. Previous reports have shown that the zebrafish and mouse Cerberus-related proteins Charon and Cerberus-like-2 (Cerl-2), respectively, act in the node region to prevent the Nodal signal from crossing to the right side, whereas chick Cerberus (cCer) has an unclear function in the left-side mesoderm. In this study, we investigate the transcriptional regulation and function of cCer in left-right development. By analyzing the enhancer activity of cCer 5' genomic sequences in electroporated chick embryos, we identified a cCer left-side enhancer that contains two FoxH1 and one SMAD binding site. We show that these Nodal-responsive elements are necessary and sufficient for the activation of transcription in the left-side mesoderm. In transgenic mouse embryos, cCer regulatory sequences behave as in chick embryos, suggesting that the cis-regulatory sequences of Cerberus-related genes have diverged during vertebrate evolution. Moreover, our findings from cCer overexpression and knockdown experiments indicate that cCer is a negative-feedback regulator of Nodal asymmetric signaling. We propose that cCer and mouse Cerl-2 have evolved distinct regulatory mechanisms but retained a conserved function in left-right development, which is to restrict Nodal activity to the left side of the embryo.

Identification of a novel role of ZIC3 in regulating cardiac development

Mutations in ZIC3 cause X-linked heterotaxy, a disorder characterized by abnormal lateralization of normally asymmetric thoracic and abdominal organs. Animal models demonstrate an early role for ZIC3 in embryonic left-right (LR) patterning. ZIC3 mutations have also been described in patients with isolated cardiovascular malformations. We wished to address the hypothesis that ZIC3 has plieotropic effects in development and may regulate cardiac development independent of its role in LR patterning. We observed significantly reduced expression of several markers of cardiac lineage commitment in Zic3(null/y) embryonic stem cells including atrial natriuretic factor (ANF), Nkx2.5 and Tbx5. Likewise, ANF expression-a molecular marker of trabecular myocardium and a direct target of multiple cardiac-specific transcription factors-was severely reduced in E9.5 Zic3 null hearts. Trabecular myocardium was reduced in these embryos. This finding was similar to that observed in embryos with cardiac-specific ablation of serum response factor (SRF), a direct transcriptional regulator of ANF expression. While ZIC3 by itself had no effect on the ANF promoter, it could bind to and inhibit a cardiac alpha-actin promoter through its zinc finger domains. We observed that ZIC3 could function as a coactivator of SRF on both cardiac alpha-actin and ANF promoters. The zinc fingers of ZIC3 and the mcm1, agamous deficiens SRF (MADS) box motif of SRF were found to mediate their physical and functional interactions. These findings reveal a novel role of ZIC3 in regulating cardiac gene expression and may explain, in part, the association of ZIC3 mutation with cardiovascular malformations.

Mice with mutations in Mahogunin ring finger‐1 (Mgrn1) exhibit abnormal patterning of the left–right axis

Mahogunin Ring Finger 1 (Mgrn1) encodes a RING-containing protein with ubiquitin ligase activity that has been implicated in pigment-type switching. In addition to having dark fur, mice lacking MGRN1 develop adult-onset spongy degeneration of the central nervous system and have reduced embryonic viability. Observation of complete situs inversus in a small proportion of adult Mgrn1 mutant mice suggested that embryonic lethality resulted from congenital heart defects due to defective establishment and/or maintenance of the left-right (LR) axis. Here we report that Mgrn1 is expressed in a pattern consistent with a role in LR patterning during early development and that many Mgrn1 mutant embryos show abnormal expression of asymmetrically expressed genes involved in LR patterning. A range of complex heart defects was observed in 20-25% of mid-to-late gestation Mgrn1 mutant embryos and another 20% were dead. This finding was consistent with 46-60% mortality of mutants by weaning age. Our results indicate that Mgrn1 acts early in the LR signaling cascade and is likely to provide new insight into this developmental process as Nodal expression was uncoupled from expression of other Nodal-responsive genes in Mgrn1 mutant embryos. Our work identifies a novel role for MGRN1 in embryonic patterning and suggests that the ubiquitination of MGRN1 target genes is essential for the proper establishment and/or maintenance of the LR axis.

Bardet–Biedl syndrome genes are important in retrograde intracellular trafficking and Kupffer's vesicle cilia function

Bardet-Biedl syndrome (BBS) is characterized by obesity, retinopathy, polydactyly, cognitive impairment, renal and cardiac anomalies as well as hypertension and diabetes. The nine known BBS genes do not appear to belong to the same functional category; yet mutation of these genes results in a nearly identical pleiotropic phenotype. Although the precise functions of the BBS proteins have yet to be determined, current data support a role in cilia function and intraflagellar transport. To gain insight into the biological processes controlled by BBS genes, we embarked on studies of six BBS orthologues from zebrafish. Knockdown of zebrafish bbs2, bbs4, bbs5, bbs6, bbs7 or bbs8 results in disruption of Kupffer's vesicle (KV), a ciliated organ thought to play a role in left-right patterning. KV defects are due to a progressive loss of cilia within the vesicle and result in subsequent alterations to organ laterality. We also note a specific defect altering retrograde melanosome transport. These studies are the first to comprehensively compare the diverse group of BBS genes in parallel and demonstrate a common role in intracellular trafficking, indicating that BBS proteins are involved in general organelle trafficking.

The roles of activin and follistatin signaling in chick gastrulation.

Activin, a TGF-beta family member, and follistatin, an activin antagonist, encode signaling proteins which have been implicated in fundamental events in early vertebrate embryogenesis, such as mesoderm and neural tissue induction, and axial patterning. In this study I examine the roles of activin and follistatin in gastrulation in the chick. Activin betaB is found to be expressed at the base of the primitive streak prior to its formation, consistent with a role in streak induction. Follistatin has a more complex and dynamic expression in Hensen's node, and exhibits a left-right (LR) asymmetry. Antagonizing endogenous activin by ectopic application of follistatin protein causes the partial dissolution of the primitive streak and node, both morphologically and as assayed by loss of expression of molecular markers. This suggests that activin is necessary for the maintenance of streak morphology, and that follistatin may be involved in termination of the anterior progress of streak growth or in suppression of supernumerary streaks. Cell ingression through the node following follistatin application is normal, suggesting that it does not depend on the pit-like morphology of the wild-type node. Finally, follistatin temporally extends the asymmetric pattern of expression of HNF3-beta, this, as well as the stronger right-sided expression of follistatin, suggests a possible role in LR patterning.