Left-right Patterning Research Articles

Klf8 regulates left-right asymmetric patterning through modulation of Kupffer\u2019s vesicle morphogenesis and spaw expression

BackgroundAlthough vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer’s vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM. This developmental step is essential to establish subsequent asymmetric organ positioning. In this study, we evaluated the role of krüppel-like factor 8 (klf8) in regulating left-right asymmetric patterning in zebrafish embryos.MethodsZebrafish klf8 expression was disrupted by both morpholino antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. Whole-mount in situ hybridization was conducted to evaluate gene expression patterns of Nodal signalling components and the positions of heart and visceral organs. Dorsal forerunner cell number was evaluated in Tg(sox17:gfp) embryos and the length and number of cilia in Kupffer’s vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody.ResultsHeart jogging, looping and visceral organ positioning were all defective in zebrafish klf8 morphants. At the 18–22 s stages, klf8 morphants showed reduced expression of genes encoding Nodal signalling components (spaw, lft1, lft2, and pitx2) in the left LPM, diencephalon, and heart. Co-injection of klf8 mRNA with klf8 morpholino partially rescued spaw expression. Furthermore, klf8 but not klf8△zf overexpressing embryos showed dysregulated bilateral expression of Nodal signalling components at late somite stages. At the 10s stage, klf8 morphants exhibited reductions in length and number of cilia in Kupffer’s vesicle, while at 75% epiboly, fewer dorsal forerunner cells were observed. Interestingly, klf8 mutant embryos, generated by a CRISPR-Cas9 system, showed bilateral spaw expression in the LPM at late somite stages. This observation may be partly attributed to compensatory upregulation of klf12b, because klf12b knockdown reduced the percentage of klf8 mutants exhibiting bilateral spaw expression.ConclusionsOur results demonstrate that zebrafish Klf8 regulates left-right asymmetric patterning by modulating both Kupffer’s vesicle morphogenesis and spaw expression in the left LPM.

An interspecies heart-to-heart: Using Xenopus to uncover the genetic basis of congenital heart disease.

Given the enormous impact congenital heart disease has on child health, it is imperative that we improve our understanding of the disease mechanisms that underlie patient phenotypes and clinical outcomes. This review will outline the merits of using the frog model, Xenopus, as a tool to study human cardiac development and left-right patterning mechanisms associated with congenital heart disease. Patient-driven gene discovery continues to provide new insight into the mechanisms of congenital heart disease, and by extension, patient phenotypes and outcomes. By identifying gene variants in CHD patients, studies in Xenopus have elucidated the molecular mechanisms of how these candidate genes affect cardiac development, both cardiogenesis as well as left-right patterning, which can have a major impact on cardiac morphogenesis. Xenopus has also proved to be a useful screening tool for the biological relevance of identified patient-mutations, and ongoing investigations continue to illuminate disease mechanisms. Analyses in model organisms can help to elucidate the disease mechanisms underlying CHD patient phenotypes. Using Xenopus to disentangle the genotype-phenotype relationships of well-known and novel disease genes could enhance the ability of physicians to efficaciously treat patients and predict clinical outcomes, ultimately improving quality of life and survival rates of patients born with congenital heart disease.

Developmental Origins of Limb Developmental Instability in Human Fetuses: Many Abnormalities Make the Difference

Fluctuating asymmetry (FA) is the small random deviation from perfect symmetry in bilateral traits and is often used to assess developmental instability (DI) experienced by organisms. In this study, with a unique dataset of 1389 deceased human fetuses, we investigated the relationship between abnormal development and human limb FA in different ways, using a more fundamental approach than usually done. We studied whether there is an underlying developmental basis of DI, as measured by FA, by investigating, first, whether limb FA can be attributed to developmental abnormalities associated with specific organ systems, germ layers or patterning processes, and second, whether limb FA increases with increasing number of developmental abnormalities either gradually, or in a threshold-like fashion. Limb FA was found to increase in fetuses with cardiovascular and nervous system abnormalities. Fetuses with ectoderm-derived abnormalities were also found to have significantly higher limb FA, but no other germ layers were found to be associated. We found no significant correlation between specific developmental processes, such as neural crest development, segmentation, midline and left-right patterning and limb FA. Although only some congenital abnormalities were correlated with limb FA, our results do suggest that limb FA increases when an increasing number of organ systems, germ layers or developmental pathways are disrupted. Therefore, we conclude that limb FA is mainly a good indicator for DI in the case of particularly severe perturbations of development and that FA does not reflect the subtler deviations from developmental stability.

Cilia and Ciliopathies in Congenital Heart Disease.

A central role for cilia in congenital heart disease (CHD) was recently identified in a large-scale mouse mutagenesis screen. Although the screen was phenotype-driven, the majority of genes recovered were cilia-related, suggesting that cilia play a central role in CHD pathogenesis. This partly reflects the role of cilia as a hub for cell signaling pathways regulating cardiovascular development. Consistent with this, many cilia-transduced cell signaling genes were also recovered, and genes regulating vesicular trafficking, a pathway essential for ciliogenesis and cell signaling. Interestingly, among CHD-cilia genes recovered, some regulate left-right patterning, indicating cardiac left-right asymmetry disturbance may play significant roles in CHD pathogenesis. Clinically, CHD patients show a high prevalence of ciliary dysfunction and show enrichment for de novo mutations in cilia-related pathways. Combined with the mouse findings, this would suggest CHD may be a new class of ciliopathy.

HCN4 ion channel function is required for early events that regulate anatomical left-right patterning in a nodal and lefty asymmetric gene expression-independent manner.

ABSTRACTLaterality is a basic characteristic of all life forms, from single cell organisms to complex plants and animals. For many metazoans, consistent left-right asymmetric patterning is essential for the correct anatomy of internal organs, such as the heart, gut, and brain; disruption of left-right asymmetry patterning leads to an important class of birth defects in human patients. Laterality functions across multiple scales, where early embryonic, subcellular and chiral cytoskeletal events are coupled with asymmetric amplification mechanisms and gene regulatory networks leading to asymmetric physical forces that ultimately result in distinct left and right anatomical organ patterning. Recent studies have suggested the existence of multiple parallel pathways regulating organ asymmetry. Here, we show that an isoform of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of ion channels (hyperpolarization-activated cyclic nucleotide-gated channel 4, HCN4) is important for correct left-right patterning. HCN4 channels are present very early in Xenopus embryos. Blocking HCN channels (Ih currents) with pharmacological inhibitors leads to errors in organ situs. This effect is only seen when HCN4 channels are blocked early (pre-stage 10) and not by a later block (post-stage 10). Injections of HCN4-DN (dominant-negative) mRNA induce left-right defects only when injected in both blastomeres no later than the 2-cell stage. Analysis of key asymmetric genes' expression showed that the sidedness of Nodal, Lefty, and Pitx2 expression is largely unchanged by HCN4 blockade, despite the randomization of subsequent organ situs, although the area of Pitx2 expression was significantly reduced. Together these data identify a novel, developmental role for HCN4 channels and reveal a new Nodal-Lefty-Pitx2 asymmetric gene expression-independent mechanism upstream of organ positioning during embryonic left-right patterning.

Roles of the cilium-associated gene CCDC11 in left-right patterning and in laterality disorders in humans.

Axial determination occurs during early stages of embryogenesis. Flaws in laterality patterning result in abnormal positioning of visceral organs, as manifested in heterotaxy syndrome, or complete left-right inversion as in situs inversus totalis. These malformations are often associated with ciliopathies, as seen in primary ciliary dyskinesia. We have recently described a novel mutation in the Coiled-Coil Domain-Containing 11 (CCDC11) gene associated with laterality disorders in a consanguineous family of Arab-Muslim origin with two affected siblings presenting with diverse phenotypes, one with heterotaxy syndrome and the other with non-primary ciliary dyskinesia situs inversus totalis. This study further characterizes the roles of CCDC11 and the implications of the identified mutation on left-right axial patterning in patient-derived cells and in the frog embryo as a model organism. We analyzed patient-derived cells and manipulated Ccdc11 levels in Xenopus laevis frog embryos. Cilia length in patient cells was longer than in controls, and CCDC11 was localized to the centriole and the actin cytoskeleton. Mutated truncated protein accumulated and was also localized to the centriole and actin cytoskeleton. In frog embryos, Ccdc11 was regulated downstream of FoxJ1, and overexpression of the full-length or truncated protein, or downregulation of the gene resulted in severe disruption of embryonic left-right axial patterning. Taken together, our initial description of the deleterious mutation in CCDC11 in patients, the current results and more recent supportive studies highlight the important role of CCDC11 in axial patterning.

The determination factors of left-right asymmetry disorders- a short review.

Laterality defects in humans, situs inversus and heterotaxy, are rare disorders, with an incidence of 1:8000 to 1:10 000 in the general population, and a multifactorial etiology. It has been proved that 1.44/10 000 of all cardiac problems are associated with malformations of left-right asymmetry and heterotaxy accounts for 3% of all congenital heart defects. It is considered that defects of situs appear due to genetic and environmental factors. Also, there is evidence that the ciliopathies (defects of structure or function) are involved in development abnormalities. Over 100 genes have been reported to be involved in left-right patterning in model organisms, but only a few are likely to candidate for left-right asymmetry defects in humans. Left-right asymmetry disorders are genetically heterogeneous and have variable manifestations (from asymptomatic to serious clinical problems). The discovery of the right mechanism of left-right development will help explain the clinical complexity and may contribute to a therapy of these disorders.

Characterization of an amphioxus heat-shock protein gene promoter and its application in vivo.

Amphioxus is a good proxy for studying the evolution and development of mechanisms during the invertebrate to vertebrate transition. However, one of the major limitations of amphioxus as a model organism is the lack of well-developed technical approaches. Promoters of genes encoding heat shock proteins are useful tools for gene function studies. In the present study, we tested heat shock temperatures on 4-cell, 128-cell, late blastula, mid-gastrula and late gastrula amphioxus embryos and optimized the heat shock conditions. We then examined the expression profile of the endogenous Hsp70 gene in embryos heat-shocked under optimum conditions. RT-qPCR revealed that the expression of Hsp70 could be induced in embryos by heat shock after the 128-cell stage, and in situ hybridization showed that the induced Hsp70 expression was predominantly detected in epidermal ectoderm. Further, we constructed two reporter gene expression plasmids carrying the Hsp70 promoter to evaluate its efficiency in vivo. Microinjection experiments showed strong red fluorescence or LacZ staining signals in injected embryos after heat shock treatment. In contrast to endogenous Hsp70, reporter gene expression was found in all three germ layers at equivalent levels. Finally, using the Hsp70 promoter, we conducted a preliminary functional analysis of two amphioxus genes, Vg1 and Cer, which play essential roles in left-right patterning in vertebrates. After heat shock at late blastula, mid-gastrula or early neurula stages, ectopic expression of Vg1 was detected in each group of treated embryos. Phenotype observation at 3-gill-slit larvae stage showed that ectopic Vg1 expression led to two-left-side or left-right reversal phenotypes, and that of Cer caused a two-right-side phenotype. Our study establishes a thermo-inducible gene expression system which will be a useful tool for gene function investigation in amphioxus embryos.

Stomach curvature is generated by left-right asymmetric gut morphogenesis.

Left-right (LR) asymmetry is a fundamental feature of internal anatomy, yet the emergence of morphological asymmetry remains one of the least understood phases of organogenesis. Asymmetric rotation of the intestine is directed by forces outside the gut, but the morphogenetic events that generate anatomical asymmetry in other regions of the digestive tract remain unknown. Here, we show in mouse and Xenopus that the mechanisms that drive the curvature of the stomach are intrinsic to the gut tube itself. The left wall of the primitive stomach expands more than the right wall, as the left epithelium becomes more polarized and undergoes radial rearrangement. These asymmetries exist across several species, and are dependent on LR patterning genes, including Foxj1, Nodal and Pitx2 Our findings have implications for how LR patterning manifests distinct types of morphological asymmetries in different contexts.

An Early Chick Embryo Culture Device for Extended Continuous Observation.

Appropriate mechanical tension of the vitelline membrane as the culture substrate for the early chick embryo is frequently reported to be required for successful in vitro development. Here we describe a modified device, made of anodized aluminum, for in vitro culture which we used for studies of left-right symmetry breaking with emphasis on morphology and gene expression as readouts. The technique allows for easy, high-throughput tissue handling and provides a suitable tension in a stable and easily reproducible manner proven to be suitable for correct molecular left-right patterning and heart looping after long-term culture.

From cytoskeletal dynamics to organ asymmetry: a nonlinear, regulative pathway underlies left-right patterning.

Consistent left-right (LR) asymmetry is a fundamental aspect of the bodyplan across phyla, and errors of laterality form an important class of human birth defects. Its molecular underpinning was first discovered as a sequential pathway of left- and right-sided gene expression that controlled positioning of the heart and visceral organs. Recent data have revised this picture in two important ways. First, the physical origin of chirality has been identified; cytoskeletal dynamics underlie the asymmetry of single-cell behaviour and patterning of the LR axis. Second, the pathway is not linear: early disruptions that alter the normal sidedness of upstream asymmetric genes do not necessarily induce defects in the laterality of the downstream genes or in organ situs Thus, the LR pathway is a unique example of two fascinating aspects of biology: the interplay of physics and genetics in establishing large-scale anatomy, and regulative (shape-homeostatic) pathways that correct molecular and anatomical errors over time. Here, we review aspects of asymmetry from its intracellular, cytoplasmic origins to the recently uncovered ability of the LR control circuitry to achieve correct gene expression and morphology despite reversals of key 'determinant' genes. We provide novel functional data, in Xenopus laevis, on conserved elements of the cytoskeleton that drive asymmetry, and comparatively analyse it together with previously published results in the field. Our new observations and meta-analysis demonstrate that despite aberrant expression of upstream regulatory genes, embryos can progressively normalize transcriptional cascades and anatomical outcomes. LR patterning can thus serve as a paradigm of how subcellular physics and gene expression cooperate to achieve developmental robustness of a body axis.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.

The balancing roles of mechanical forces during left-right patterning and asymmetric morphogenesis.

Left-right patterning and asymmetric morphogenesis arise from a complex set of molecular and cellular interactions that are particularly dynamic and associated with mechanical forces. How do mechanical forces translate into tissular asymmetries? Are these forces asymmetrical de novo, or do they build up from pre-existing asymmetries? Advances in developmental genetics, live imaging and cell biology have recently shed light on the origins of mechanical forces generated at the cell scale and their implication in asymmetric patterning and morphogenesis is now emerging. Here we ask when and how, molecular asymmetries and mechanical forces contribute to left-right patterning and organ asymmetries.

Congenital Heart Disease Genetics Uncovers Context-Dependent Organization and Function of Nucleoporins at Cilia

Human genomics is identifying candidate genes for congenital heart disease (CHD), but discovering the underlying mechanisms remains challenging. In a patient with CHD and heterotaxy (Htx), a disorder of left-right patterning, we previously identified a duplication in Nup188. However, a mechanism to explain how a component of the nuclear pore complex (NPC) could cause Htx/CHD was undefined. Here, we show that knockdown of Nup188 or its binding partner Nup93 leads to a loss of cilia during embryonic development while leaving NPC function largely intact. Many data, including the localization of endogenous Nup188/93 at cilia bases, support theirdirect role at cilia. Super-resolution imaging ofNup188 shows two barrel-like structures with dimensions and organization incompatible with an NPC-like ring, arguing against a proposed "ciliary pore complex." We suggest that the nanoscale organization and function of nucleoporins are context dependent in a way that is required for the structure of the heart.

Gap junctional signaling in pattern regulation: Physiological network connectivity instructs growth and form.

Gap junctions (GJs) are aqueous channels that allow cells to communicate via physiological signals directly. The role of gap junctional connectivity in determining single-cell functions has long been recognized. However, GJs have another important role: the regulation of large-scale anatomical pattern. GJs are not only versatile computational elements that allow cells to control which small molecule signals they receive and emit, but also establish connectivity patterns within large groups of cells. By dynamically regulating the topology of bioelectric networks in vivo, GJs underlie the ability of many tissues to implement complex morphogenesis. Here, a review of recent data on patterning roles of GJs in growth of the zebrafish fin, the establishment of left-right patterning, the developmental dysregulation known as cancer, and the control of large-scale head-tail polarity, and head shape in planarian regeneration has been reported. A perspective in which GJs are not only molecular features functioning in single cells, but also enable global neural-like dynamics in non-neural somatic tissues has been proposed. This view suggests a rich program of future work which capitalizes on the rapid advances in the biophysics of GJs to exploit GJ-mediated global dynamics for applications in birth defects, regenerative medicine, and morphogenetic bioengineering. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 643-673, 2017.

Mutations in zebrafish pitx2 model congenital malformations in Axenfeld-Rieger syndrome but do not disrupt left-right placement of visceral organs

Pitx2 is a conserved homeodomain transcription factor that has multiple functions during embryonic development. Mutations in human PITX2 cause autosomal dominant Axenfeld-Rieger syndrome (ARS), characterized by congenital eye and tooth malformations. Pitx2−/− knockout mouse models recapitulate aspects of ARS, but are embryonic lethal. To date, ARS treatments remain limited to managing individual symptoms due to an incomplete understanding of PITX2 function. In addition to regulating eye and tooth development, Pitx2 is a target of a conserved Nodal (TGFβ) signaling pathway that mediates left-right (LR) asymmetry of visceral organs. Based on its highly conserved asymmetric expression domain, the Nodal-Pitx2 axis has long been considered a common denominator of LR development in vertebrate embryos. However, functions of Pitx2 during asymmetric organ morphogenesis are not well understood. To gain new insight into Pitx2 function we used genome editing to create mutations in the zebrafish pitx2 gene. Mutations in the pitx2 homeodomain caused phenotypes reminiscent of ARS, including aberrant development of the cornea and anterior chamber of the eye and reduced or absent teeth. Intriguingly, LR asymmetric looping of the heart and gut was normal in pitx2 mutants. These results suggest conserved roles for Pitx2 in eye and tooth development and indicate Pitx2 is not required for asymmetric looping of zebrafish visceral organs. This work establishes zebrafish pitx2 mutants as a new animal model for investigating mechanisms underlying congenital malformations in ARS and high-throughput drug screening for ARS therapeutics. Additionally, pitx2 mutants present a unique opportunity to identify new genes involved in vertebrate LR patterning. We show Nodal signaling—independent of Pitx2—controls asymmetric expression of the fatty acid elongase elovl6 in zebrafish, pointing to a potential novel pathway during LR organogenesis.

Misexpression of Tbx18 in cardiac chambers of fetal mice interferes with chamber-specific developmental programs but does not induce a pacemaker-like gene signature

Initiation of cardiac excitation depends on a specialized group of cardiomyocytes at the venous pole of the heart, the sinoatrial node (SAN). The T-box transcription factor gene Tbx18 is expressed in the SAN myocardium and is required for formation of a large portion of the pacemaker. Previous studies suggested that Tbx18 is also sufficient to reprogram ventricular cardiomyocytes into SAN cells in rat, guinea-pig and pig hearts. To evaluate the consequences of misexpression of Tbx18 for imposing a nodal phenotype onto chamber myocardial cells in fetal mice, we used two independent conditional approaches with chamber-specific cre driver lines and an HprtTbx18 misexpression allele. Myh6-Cre/+;HprtTbx18/y mice developed dilated atria with thickened walls, reduced right ventricles and septal defects that resulted in reduced embryonic and post-natal survival. Tagln-Cre/+;HprtTbx18/y mice exhibited slightly smaller hearts with rounded trabeculae that supported normal embryonic survival. Molecular analyses showed that the SAN gap junction and ion channel profile was not ectopically induced in chamber myocardium but the working myocardial gene program was partially inhibited in atria and ventricles of both misexpression models. Left atrial expression of Pitx2 was strongly repressed in Myh6-Cre/+;HprtTbx18/y embryos. We conclude that exclusion of Tbx18 expression from the developing atria and (right) ventricle is important to achieve normal cardiac left-right patterning and myocardial differentiation, and that Tbx18 is not sufficient to induce full SAN differentiation of chamber cardiomyocytes in fetal mice.

Prickle3 synergizes with Wtip to regulate basal body organization and cilia growth.

PCP proteins maintain planar polarity in many epithelial tissues and have been implicated in cilia development in vertebrate embryos. In this study we examine Prickle3 (Pk3), a vertebrate homologue of Drosophila Prickle, in Xenopus gastrocoel roof plate (GRP). GRP is a tissue equivalent to the mouse node, in which cilia-generated flow promotes left-right patterning. We show that Pk3 is enriched at the basal body of GRP cells but is recruited by Vangl2 to anterior cell borders. Interference with Pk3 function disrupted the anterior polarization of endogenous Vangl2 and the posterior localization of cilia in GRP cells, demonstrating its role in PCP. Strikingly, in cells with reduced Pk3 activity, cilia growth was inhibited and γ-tubulin and Nedd1 no longer associated with the basal body, suggesting that Pk3 has a novel function in basal body organization. Mechanistically, this function of Pk3 may involve Wilms tumor protein 1-interacting protein (Wtip), which physically associates with and cooperates with Pk3 to regulate ciliogenesis. We propose that, in addition to cell polarity, PCP components control basal body organization and function.

Conserved roles for cytoskeletal components in determining laterality.

Consistently-biased left-right (LR) patterning is required for the proper placement of organs including the heart and viscera. The LR axis is especially fascinating as an example of multi-scale pattern formation, since here chiral events at the subcellular level are integrated and amplified into asymmetric transcriptional cascades and ultimately into the anatomical patterning of the entire body. In contrast to the other two body axes, there is considerable controversy about the earliest mechanisms of embryonic laterality. Many molecular components of asymmetry have not been widely tested among phyla with diverse bodyplans, and it is unknown whether parallel (redundant) pathways may exist that could reverse abnormal asymmetry states at specific checkpoints in development. To address conservation of the early steps of LR patterning, we used the Xenopus laevis (frog) embryo to functionally test a number of protein targets known to direct asymmetry in plants, fruit fly, and rodent. Using the same reagents that randomize asymmetry in Arabidopsis, Drosophila, and mouse embryos, we show that manipulation of the microtubule and actin cytoskeleton immediately post-fertilization, but not later, results in laterality defects in Xenopus embryos. Moreover, we observed organ-specific randomization effects and a striking dissociation of organ situs from effects on the expression of left side control genes, which parallel data from Drosophila and mouse. Remarkably, some early manipulations that disrupt laterality of transcriptional asymmetry determinants can be subsequently "rescued" by the embryo, resulting in normal organ situs. These data reveal the existence of novel corrective mechanisms, demonstrate that asymmetric expression of Nodal is not a definitive marker of laterality, and suggest the existence of amplification pathways that connect early cytoskeletal processes to control of organ situs bypassing Nodal. Counter to alternative models of symmetry breaking during neurulation (via ciliary structures absent in many phyla), our data suggest a widely-conserved role for the cytoskeleton in regulating left-right axis formation immediately after fertilization of the egg. The novel mechanisms that rescue organ situs, even after incorrect expression of genes previously considered to be left-side master regulators, suggest LR patterning as a new context in which to explore multi-scale redundancy and integration of patterning from the subcellular structure to the entire bodyplan.

DNAH6 and Its Interactions with PCD Genes in Heterotaxy and Primary Ciliary Dyskinesia.

Heterotaxy, a birth defect involving left-right patterning defects, and primary ciliary dyskinesia (PCD), a sinopulmonary disease with dyskinetic/immotile cilia in the airway are seemingly disparate diseases. However, they have an overlapping genetic etiology involving mutations in cilia genes, a reflection of the common requirement for motile cilia in left-right patterning and airway clearance. While PCD is a monogenic recessive disorder, heterotaxy has a more complex, largely non-monogenic etiology. In this study, we show mutations in the novel dynein gene DNAH6 can cause heterotaxy and ciliary dysfunction similar to PCD. We provide the first evidence that trans-heterozygous interactions between DNAH6 and other PCD genes potentially can cause heterotaxy. DNAH6 was initially identified as a candidate heterotaxy/PCD gene by filtering exome-sequencing data from 25 heterotaxy patients stratified by whether they have airway motile cilia defects. dnah6 morpholino knockdown in zebrafish disrupted motile cilia in Kupffer’s vesicle required for left-right patterning and caused heterotaxy with abnormal cardiac/gut looping. Similarly DNAH6 shRNA knockdown disrupted motile cilia in human and mouse respiratory epithelia. Notably a heterotaxy patient harboring heterozygous DNAH6 mutation was identified to also carry a rare heterozygous PCD-causing DNAI1 mutation, suggesting a DNAH6/DNAI1 trans-heterozygous interaction. Furthermore, sequencing of 149 additional heterotaxy patients showed 5 of 6 patients with heterozygous DNAH6 mutations also had heterozygous mutations in DNAH5 or other PCD genes. We functionally assayed for DNAH6/DNAH5 and DNAH6/DNAI1 trans-heterozygous interactions using subthreshold double-morpholino knockdown in zebrafish and showed this caused heterotaxy. Similarly, subthreshold siRNA knockdown of Dnah6 in heterozygous Dnah5 or Dnai1 mutant mouse respiratory epithelia disrupted motile cilia function. Together, these findings support an oligogenic disease model with broad relevance for further interrogating the genetic etiology of human ciliopathies.

Paraxial Nodal Expression Reveals a Novel Conserved Structure of the Left-Right Organizer in Four Mammalian Species

Nodal activity in the left lateral plate mesoderm is a conserved sign of irreversible left-right asymmetry at early somite stages of the vertebrate embryo. An earlier, paraxial nodal domain accompanies the emergence and initial extension of the notochord and is either left-sided, as in the chick and pig, or symmetrical, as in the mouse and rabbit; intriguingly, this interspecific dichotomy is mirrored by divergent morphological features of the posterior notochord (also known as the left-right organizer), which is ventrally exposed to the yolk sac cavity and carries motile cilia in the latter 2 species only. By introducing the cattle embryo as a new model organism for early left-right patterning, we present data to establish 2 groups of mammals characterized by both the morphology of the left-right organizer and the dynamics of paraxial nodal expression: presence and absence of a ventrally open surface of the early (plate-like) posterior notochord correlates with a symmetrical (in mice and rabbits) versus an asymmetrical (in pigs and cattle) paraxial nodal expression domain next to the notochordal plate. High-resolution histological analysis reveals that the latter domain defines in all 4 mammals a novel ‘parachordal' axial mesoderm compartment, the topography of which changes according to the specific regression of the similarly novel subchordal mesoderm during the initial phases of notochord development. In conclusion, the mammalian axial mesoderm compartment (1) shares critical conserved features despite the marked differences in early notochord morphology and early left-right patterning and (2) provides a dynamic topographical framework for nodal activity as part of the mammalian left-right organizer.