Embryonic Node Research Articles

Establishment of Cardiac Laterality.

Formation of the vertebrate heart with its complex arterial and venous connections is critically dependent on patterning of the left-right axis during early embryonic development. Abnormalities in left-right patterning can lead to a variety of complex life-threatening congenital heart defects. A highly conserved pathway responsible for left-right axis specification has been uncovered. This pathway involves initial asymmetric activation of a nodal signaling cascade at the embryonic node, followed by its propagation to the left lateral plate mesoderm and activation of left-sided expression of the Pitx2 transcription factor specifying visceral organ asymmetry. Intriguingly, recent work suggests that cardiac laterality is encoded by intrinsic cell and tissue chirality independent of Nodal signaling. Thus, Nodal signaling may be superimposed on this intrinsic chirality, providing additional instructive cues to pattern cardiac situs. The impact of intrinsic chirality and the perturbation of left-right patterning on myofiber organization and cardiac function warrants further investigation. We summarize recent insights gained from studies in animal models and also some human clinical studies in a brief overview of the complex processes regulating cardiac asymmetry and their impact on cardiac function and the pathogenesis of congenital heart defects.

Single cell RNA analysis of the left–right organizer transcriptome reveals potential novel heterotaxy genes

The establishment of left–right patterning in mice occurs at a transient structure called the embryonic node or left–right organizer (LRO). Previous analysis of the LRO has proven challenging due to the small cell number and transient nature of this structure. Here, we seek to overcome these difficulties to define the transcriptome of the LRO. Specifically, we used single cell RNA sequencing of 0–1 somite embryos to identify LRO enriched genes which were compared to bulk RNA sequencing of LRO cells isolated by fluorescent activated cell sorting. Gene ontology analysis indicated an enrichment of genes associated with cilia and laterality terms. Furthermore, comparison to previously identified LRO genes identified 127 novel LRO genes, including Ttll3, Syne1 and Sparcl1, for which the expression patterns were validated using whole mount in situ hybridization. This list of novel LRO genes will be a useful resource for further studies on LRO morphogenesis, the establishment of laterality and the genetic causes of heterotaxy.

Development's Inaugural Outstanding Paper Prize.

Development's Inaugural Outstanding Paper Prize.

Layered origins of lymphoid tissue inducer cells.

The Innate Lymphoid Cell (ILC) family is a relatively recently described immune cell family involved in innate immune responses and tissue homeostasis. Lymphoid Tissue Inducer (LTi) cells are part of the type 3 (ILC3) family. The ILC3 family is the main ILC population within the embryo, in which the LTi cells are critically associated with embryonic lymph node formation. Recent studies have shown more insights in ILC origin and residency from local embryonic and tissue resident precursors. Embryonic LTi cells originating from a different hemogenic endothelial source were shown to be replaced by HSC derived progenitors in adult. This review will discuss the layered origin of the ILC3 family with an emphasis on the LTi cell lineage.

Biophysical Analysis of Mechanical Signals in Immotile Cilia of Mouse Embryonic Nodes Using Advanced Microscopic Techniques.

Immotile cilia of crown cells at the node of mouse embryos are required for sensing leftward fluid flow that gives rise to the breaking of left-right (L-R) symmetry. The flow-sensing mechanism has long remained elusive, mainly because of difficulties inherent in manipulating and precisely analyzing the cilium. Recent progress in optical microscopy and biophysical analysis has allowed us to study the mechanical signals involving primary cilia. In this study, we used high-resolution imaging with mechanical modeling to assess the membrane tension in a single cilium. Optical tweezers, a technique used to trap sub-micron-sized particles with a highly focused laser beam, allowed us to manipulate individual cilia. Super-resolution microscopy allowed us to discern the precise localization of ciliary proteins. Using this protocol, we provide a method for applying these techniques to cilia in mouse embryonic nodes. This method is widely applicable to the determination of mechanical signals in other cilia.

Complex wave’s impact on the cilia-generated flow in a vertical tube with an endoscope

ABSTRACT This research object is focused to discuss the cilia-driven flow along complex wave progression via cilia tips. Suspension of nanocomposites is considered in this analysis to discuss the enhanced heat transfer rate properties of the base fluid blood. The endoscopic tube is inserted into the flow region covered by cilia tips to overcome the cilia disorders. Governed mathematical equations are solved by the Mathematica DSolve command after using lubrication approximation. To see the theoretical consequence of complex cilia movement along the tube, graphical results are plotted for different innovative parameters which are discussed in detail. Streamlines are strategized to see the blood circulation pattern in the presence of the complex cilia waves. From a theoretical point of view, it is noted that complex cilia waves accelerate the fluid circulations more significantly near the ciliated walls. Moreover, the cilia-driven fluid flow has a substantial role in multiple progressions such as clearance of respiratory mucus, right-left pattern in the embryonic node, gamete transport in the oviduct, and cerebrospinal fluid flow.

Sequential action of JNK genes establishes the embryonic left-right axis.

ABSTRACTThe establishment of the left-right axis is crucial for the placement, morphogenesis and function of internal organs. Left-right specification is proposed to be dependent on cilia-driven fluid flow in the embryonic node. Planar cell polarity (PCP) signalling is crucial for patterning of nodal cilia, yet downstream effectors driving this process remain elusive. We have examined the role of the JNK gene family, a proposed downstream component of PCP signalling, in the development and function of the zebrafish node. We show jnk1 and jnk2 specify length of nodal cilia, generate flow in the node and restrict southpaw to the left lateral plate mesoderm. Moreover, loss of asymmetric southpaw expression does not result in disturbances to asymmetric organ placement, supporting a model in which nodal flow may be dispensable for organ laterality. Later, jnk3 is required to restrict pitx2c expression to the left side and permit correct endodermal organ placement. This work uncovers multiple roles for the JNK gene family acting at different points during left-right axis establishment. It highlights extensive redundancy and indicates JNK activity is distinct from the PCP signalling pathway.

The embryonic node behaves as an instructive stem cell niche for axial elongation

In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen's node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle-related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.

Vangl2-environment interaction causes severe neural tube defects, without abnormal neuroepithelial convergent extension.

ABSTRACTPlanar cell polarity (PCP) signalling is vital for initiation of mouse neurulation, with diminished convergent extension (CE) cell movements leading to craniorachischisis, a severe neural tube defect (NTD). Some humans with NTDs also have PCP gene mutations but these are heterozygous, not homozygous as in mice. Other genetic or environmental factors may interact with partial loss of PCP function in human NTDs. We found that reduced sulfation of glycosaminoglycans interacts with heterozygosity for the Lp allele of Vangl2 (a core PCP gene), to cause craniorachischisis in cultured mouse embryos, with rescue by exogenous sulphate. We hypothesized that this glycosaminoglycan–PCP interaction may regulate CE, but, surprisingly, DiO labelling of the embryonic node demonstrates no abnormality of midline axial extension in sulfation-depleted Lp/+ embryos. Positive-control Lp/Lp embryos show severe CE defects. Abnormalities were detected in the size and shape of somites that flank the closing neural tube in sulfation-depleted Lp/+ embryos. We conclude that failure of closure initiation can arise by a mechanism other than faulty neuroepithelial CE, with possible involvement of matrix-mediated somite expansion, adjacent to the closing neural tube.

Loss of Zic3 impairs planar cell polarity leading to abnormal left-right signaling, heart defects and neural tube defects.

Loss of function of ZIC3 causes heterotaxy (OMIM #306955), a disorder characterized by organ laterality defects including complex heart defects. Studies using Zic3 mutant mice have demonstrated that loss of Zic3 causes heterotaxy due to defects in establishment of left-right (LR) signaling, but the mechanistic basis for these defects remains unknown. Here, we demonstrate Zic3 null mice undergo cilia positioning defects at the embryonic node consistent with impaired planar cell polarity (PCP). Cell-based assays demonstrate that ZIC3 must enter the nucleus to regulate PCP and identify multiple critical ZIC3 domains required for regulation of PCP signaling. Furthermore, we show that Zic3 displays a genetic interaction with the PCP membrane protein Vangl2 and the PCP effector genes Rac1 and Daam1 resulting in increased frequency and severity of neural tube and heart defects. Gene and protein expression analyses indicate that Zic3 null embryos display disrupted expression of PCP components and reduced phosphorylation of the core PCP protein DVL2 at the time of LR axis determination. These results demonstrate that ZIC3 interacts with PCP signaling during early development, identifying a novel role for this transcription factor, and adding additional evidence about the importance of PCP function for normal LR patterning and subsequent heart development.

Embryonic protein NODAL regulates the breast tumor microenvironment by reprogramming cancer-derived secretomes

The tumor microenvironment (TME) is an important mediator of breast cancer progression. Cancer-associated fibroblasts constitute a major component of the TME and may originate from tissue-associated fibroblasts or infiltrating mesenchymal stromal cells (MSCs). The mechanisms by which cancer cells activate fibroblasts and recruit MSCs to the TME are largely unknown, but likely include deposition of a pro-tumorigenic secretome. The secreted embryonic protein NODAL is clinically associated with breast cancer stage and promotes tumor growth, metastasis, and vascularization. Herein, we show that NODAL expression correlates with the presence of activated fibroblasts in human triple-negative breast cancers and that it directly induces Cancer-associated fibroblasts phenotypes. We further show that NODAL reprograms cancer cell secretomes by simultaneously altering levels of chemokines (e.g., CXCL1), cytokines (e.g., IL-6) and growth factors (e.g., PDGFRA), leading to alterations in MSC chemotaxis. We therefore demonstrate a hitherto unappreciated mechanism underlying the dynamic regulation of the TME.

Centrosomal protein FOR20 knockout mice display embryonic lethality and left-right patterning defects.

Centrosomal protein FOR20 has been reported to be crucial for essential cellular processes, including ciliogenesis, cell migration, and cell cycle in vertebrates. However, the function of FOR20 during mammalian embryonic development remains unknown. To investigate the invivo function of the For20 gene in mammals, we generated For20 homozygous knockout mice by gene targeting. Our data reveal that homozygous knockout of For20 results in significant embryonic growth arrest and lethality during gestation, while the heterozygotes show no obvious defects. The absence of For20 leads to impaired left-right patterning of embryos and reduced cilia in the embryonic node. Deletion of For20 also disrupts angiogenesis in yolk sacs and embryos. These results highlight a critical role of For20 in early mammalian embryogenesis.

Abstract PO028: Embryonic protein NODAL as a potential modulator of the tumour microenvironment: Breast cancer secretome reprogramming and fibroblast activation

Abstract The tumour microenvironment (TME), consisting of stromal cell types including fibroblasts, endothelial, immune and mesenchymal stromal cells (MSCs), is an important mediator of breast cancer (BCa) progression. BCa cells regulate the composition of the TME by secreting a myriad of factors; however, mechanisms governing the dynamic reciprocity between BCa and TME components are largely unknown. Cancer-associated fibroblasts (CAFs) are a major stromal component in many BCa and have been shown to be activated by BCa cells. MSCs recruited to the TME are an important source of CAFs in many cancers; therefore, characterizing the factors involved in MSC recruitment could provide additional avenues for therapeutic intervention by disrupting the TME in BCa. NODAL, an embryonic morphogen belonging to the TGF-beta superfamily, is clinically associated with BCa progression, and has been shown to sustain BCa stem cells and to promote tumour growth, metastasis, and vascularization. Herein, we report that NODAL expression positively correlates with the presence of CAFs in the stroma of triple-negative human BCa tissues, as determined by NODAL and a-smooth muscle Actin (SMA) staining. Further, our in vitro studies show that NODAL directly induces primary fibroblast activation and chemotaxis. Proteomic analysis of the conditioned media of NODAL-overexpressing MDA-MB-231 and SUM149 BCa cells by mass spectrometry reveal that NODAL reprograms BCa secretomes by simultaneously altering levels of chemokines (e.g. CXCL1), growth factors (e.g. PDGFRA) and cytokines (e.g. IL-6), and that the NODAL-regulated secretome, but not NODAL directly, impacts the ability of BCa cells to affect MSC chemotaxis. We provide evidence that IL-6 promotes MSC chemotaxis, and further discuss the context-dependent effects of NODAL in BCa cell lines. Collectively, our results suggest that factors present in NODAL-regulated secretomes may induce MSC recruitment to the breast TME, where they may contribute to CAF population, therefore demonstrating a hitherto unappreciated role of NODAL as a dynamic regulator of breast TME components. Citation Format: Dylan Dieters-Castator, Paola M. Dantonio, Matt Piaseczny, Guihua Zhang, Jiahui Liu, Miljan Kuljanin, Stephen Sherman, Michael Jewer, Katherine Quesnel, Eun-Young Kang, Martin Köbel, Gabrielle M. Siegers, Andrew Leask, David Hess, Gilles Lajoie, Lynne-Marie Postovit. Embryonic protein NODAL as a potential modulator of the tumour microenvironment: Breast cancer secretome reprogramming and fibroblast activation [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO028.

Identification of a CCDC114 variant in a Han-Chinese patient with situs inversus.

The function and position of the internal organs within the human body are based on left-right (LR) asymmetry. Human LR asymmetry disorders are characterized by abnormal LR asymmetric arrangement of the internal organs resulting from defective embryonic nodal cilia and nodal signaling pathway. The coiled-coil domain containing 114 gene (CCDC114) is related to the biogenesis of cilia and attachment of the outer dynein arms (ODAs) to the axoneme of cilia. Mutations in the CCDC114 gene are reported to cause a subtype of primary ciliary dyskinesia (PCD) named ciliary dyskinesia, primary, 20 (CILD20). Patients with CCDC114 mutations present with a type of ciliopathy with high clinical heterogeneity. In the present study, a Han-Chinese patient with situs inversus was recruited. Exome sequencing was performed on this patient combined with variant validation by Sanger sequencing. A homozygous variant c.584T>C (p.L195P) in the CCDC114 gene was identified as the likely genetic cause for situs inversus in this patient. The findings of our study extend the mutational spectrum of the CCDC114 gene, and contribute to clarifying the pathogenesis of human ciliopathies and benefit genetic counseling.

Hedgehog–FGF signaling axis patterns anterior mesoderm during gastrulation

The mechanisms used by embryos to pattern tissues across their axes has fascinated developmental biologists since the founding of embryology. Here, using single-cell technology, we interrogate complex patterning defects and define a Hedgehog (Hh)-fibroblast growth factor (FGF) signaling axis required for anterior mesoderm lineage development during gastrulation. Single-cell transcriptome analysis of Hh-deficient mesoderm revealed selective deficits in anterior mesoderm populations, culminating in defects to anterior embryonic structures, including the pharyngeal arches, heart, and anterior somites. Transcriptional profiling of Hh-deficient mesoderm during gastrulation revealed disruptions to both transcriptional patterning of the mesoderm and FGF signaling for mesoderm migration. Mesoderm-specific Fgf4/Fgf8 double-mutants recapitulated anterior mesoderm defects and Hh-dependent GLI transcription factors modulated enhancers at FGF gene loci. Cellular migration defects during gastrulation induced by Hh pathway antagonism were mitigated by the addition of FGF4 protein. These findings implicate a multicomponent signaling hierarchy activated by Hh ligands from the embryonic node and executed by FGF signals in nascent mesoderm to control anterior mesoderm patterning.

Aberrantly Expressed Embryonic Protein NODAL Alters Breast Cancer Cell Susceptibility to γδ T Cell Cytotoxicity.

Gamma delta (γδ) T cells kill transformed cells, and increased circulating γδ T cells levels correlate with improved outcome in cancer patients; however, their function within the breast tumor microenvironment (TME) remains controversial. As tumors progress, they begin to express stem-cell associated proteins, concomitant with the emergence of therapy resistant metastatic disease. For example, invasive breast cancers often secrete the embryonic morphogen, NODAL. NODAL has been shown to promote angiogenesis, therapy resistance and metastasis in breast cancers. However, to date, little is known about how this secreted protein may interact with cells in the TME. Herein we explore how NODAL in the TME may influence γδ T cell function. We have assessed the proximity of γδ T cells to NODAL in a cohort of triple negative breast tumors. In all cases in which γδ T cells could be identified in these tumors, γδ T cells were found in close proximity to NODAL-expressing tumor cells. Migration of γδ and αβ T cells was similar toward MDA-MB-231 cells in which NODAL had been knocked down (shN) and MDA-MB-231 scrambled control cells (shC). Furthermore, Vδ1 γδ T cells did not migrate preferentially toward conditioned medium from these cell lines. While 24-h exposure to NODAL did not impact CD69, PD-1, or T cell antigen receptor (TCR) expression on γδ T cells, long term exposure resulted in decreased Vδ2 TCR expression. Maturation of γδ T cells was not significantly influenced by NODAL stimulation. While neither short- nor long-term NODAL stimulation impacted the ability of γδ T cells to kill MCF-7 breast cancer cells, the absence of NODAL resulted in greater sensitivity of targets to γδ T cell cytotoxicity, while overexpression of NODAL conferred resistance. This appeared to be at least in part due to an inverse correlation between NODAL and surface MICA/B expression on breast cancer target lines. As such, it appears that NODAL may play a role in strategies employed by breast cancer cells to evade γδ T cell targeting, and this should be considered in the development of safe and effective γδ T cell immunotherapies.

Genetically Modified Porcine Mesenchymal Stem Cells by Lentiviral Tbx18 Create a Biological Pacemaker.

Background Tbx18 is a vital transcription factor involved in embryonic sinoatrial node (SAN) formation process but is gradually vanished after birth. Myocardial injection of lentiviral Tbx18 converts cardiomyocytes into pacemaker-like cells morphologically and functionally. In this in vitro and in vivo study, genetical modification of porcine bone mesenchymal stem cells (BMSCs) by recapturing the Tbx18 expression creates a biological pacemaker which was examined. Methods The isolated porcine BMSCs were transfected with lentiviral Tbx18, and the induced pacemaker-like cells were analyzed using real-time polymerase chain reaction and western blotting to investigate the efficiency of transformation. Then, the induced pacemaker-like cells were implanted into the right ventricle of the SAN dysfunction porcine model after the differentiation process. Biological pacemaker activity and ectopic pacing region were tested by an electrocardiograph (ECG) monitor. Results The isolated porcine BMSCs expressed specific surface markers of stem cells; meanwhile, the expression of myocardial markers was upregulated significantly after lentiviral Tbx18 transfection. The porcine SAN dysfunction model was constructed by electrocoagulation using a surgical electrotome. The results showed that the mean heart beat (HR) of BMSCs-Tbx18 was significantly higher than that of BMSCs-GFP. An ectopic pacing region was affirmed into the right ventricle by ECG after implantation of BMSCs-Tbx18. Conclusion It was verified that Lenti-Tbx18 is capable of transducing porcine BMSCs into pacemaker-like cells. Genetically modified porcine BMSCs by lentiviral Tbx18 could create a biological pacemaker. However, further researches in large-scale animals are required to rule out unexpected complications prior to application in clinical practice.

New insights into ion channel-dependent signalling during left-right patterning.

The left-right organizer (LRO) in the mouse consists of pit cells within the depression, located at the end of the developing notochord, also known as the embryonic node and crown cells lining the outer periphery of the node. Cilia on pit cells are posteriorly tilted, rotate clockwise and generate leftward fluid flow. Primary cilia on crown cells are required to interpret the directionality of fluid movement and initiate flow-dependent gene transcription. Crown cells express PC1-L1 and PC2, which may form a heteromeric polycystin channel complex on primary cilia. It is still only poorly understood how fluid flow activates the ciliary polycystin complex. Besides polycystin channels voltage gated channels like HCN4 and KCNQ1 have been implicated in establishing asymmetry. How this electrical network of ion channels initiates left-sided signalling cascades and differential gene expression is currently only poorly defined.

Survey of the Ciliary Motility Machinery of Drosophila Sperm and Ciliated Mechanosensory Neurons Reveals Unexpected Cell-Type Specific Variations: A Model for Motile Ciliopathies.

The motile cilium/flagellum is an ancient eukaryotic organelle. The molecular machinery of ciliary motility comprises a variety of cilium-specific dynein motor complexes along with other complexes that regulate their activity. Assembling the motors requires the function of dedicated “assembly factors” and transport processes. In humans, mutation of any one of at least 40 different genes encoding components of the motility apparatus causes Primary Ciliary Dyskinesia (PCD), a disease of defective ciliary motility. Recently, Drosophila has emerged as a model for motile cilia biology and motile ciliopathies. This is somewhat surprising as most Drosophila cells lack cilia, and motile cilia are confined to just two specialized cell types: the sperm flagellum with a 9+2 axoneme and the ciliated dendrite of auditory/proprioceptive (chordotonal, Ch) neurons with a 9+0 axoneme. To determine the utility of Drosophila as a model for motile cilia, we survey the Drosophila genome for ciliary motility gene homologs, and assess their expression and function. We find that the molecules of cilium motility are well conserved in Drosophila. Most are readily characterized by their restricted cell-type specific expression patterns and phenotypes. There are also striking differences between the two motile ciliated cell types. Notably, sperm and Ch neuron cilia express and require entirely different outer dynein arm variants—the first time this has been clearly established in any organism. These differences might reflect the specialized functions for motility in the two cilium types. Moreover, the Ch neuron cilia lack the critical two-headed inner arm dynein (I1/f) but surprisingly retain key regulatory proteins previously associated with it. This may have implications for other motile 9+0 cilia, including vertebrate embryonic nodal cilia required for left-right axis asymmetry. We discuss the possibility that cell-type specificity in ciliary motility machinery might occur in humans, and therefore underlie some of the phenotypic variation observed in PCD caused by different gene mutations. Our work lays the foundation for the increasing use of Drosophila as an excellent model for new motile ciliary gene discovery and validation, for understanding motile cilium function and assembly, as well as understanding the nature of genetic defects underlying human motile ciliopathies.

Recessive DNAH9 Loss-of-Function Mutations Cause Laterality Defects and Subtle Respiratory Ciliary-Beating Defects

Dysfunction of motile monocilia, altering the leftward flow at the embryonic node essential for determination of left-right body asymmetry, is a major cause of laterality defects. Laterality defects are also often associated with reduced mucociliary clearance caused by defective multiple motile cilia of the airway and are responsible for destructive airway disease. Outer dynein arms (ODAs) are essential for ciliary beat generation, and human respiratory cilia contain different ODA heavy chains (HCs): the panaxonemally distributed γ-HC DNAH5, proximally located β-HC DNAH11 (defining ODA type 1), and the distally localized β-HC DNAH9 (defining ODA type 2). Here we report loss-of-function mutations in DNAH9 in five independent families causing situs abnormalities associated with subtle respiratory ciliary dysfunction. Consistent with the observed subtle respiratory phenotype, high-speed video microscopy demonstrates distally impaired ciliary bending in DNAH9 mutant respiratory cilia. DNAH9-deficient cilia also lack other ODA components such as DNAH5, DNAI1, and DNAI2 from the distal axonemal compartment, demonstrating an essential role of DNAH9 for distal axonemal assembly of ODAs type 2. Yeast two-hybrid and co-immunoprecipitation analyses indicate interaction of DNAH9 with the ODA components DNAH5 and DNAI2 as well as the ODA-docking complex component CCDC114. We further show that during ciliogenesis of respiratory cilia, first proximally located DNAH11 and then distally located DNAH9 is assembled in the axoneme. We propose that the β-HC paralogs DNAH9 and DNAH11 achieved specific functional roles for the distinct axonemal compartments during evolution with human DNAH9 function matching that of ancient β-HCs such as that of the unicellular Chlamydomonas reinhardtii.