The development and evolution of left‐right asymmetry in invertebrates: Lessons from Drosophila and snails

The unique nature of body handedness, which is distinct from the anteroposterior and dorsoventral polarities, has been attracting growing interest in diverse biological disciplines. Recent research progress on the left‐right asymmetry of animal development has focused new attention on the mechanisms underlying the development and evolution of invertebrate handedness. This exploratory review of currently available information illuminates the prospective value of Drosophila and pulmonate snails for innovative new research aimed at elucidating these mechanisms. For example, findings in Drosophila and snails suggest that an actin filament–dependent mechanism may be evolutionarily conserved in protostomes. The polarity conservation of primary asymmetry across most metazoan phyla, which visceral handedness represents, indicates developmental constraint and purifying selection as possible but unexplored mechanisms. Comparative studies using Drosophila and snails, which have the great advantages of using genetic and evolutionary approaches, will accelerate our understanding of the mechanisms governing the conservation and diversity of animal handedness. Developmental Dynamics 237:3497–3515, 2008. © 2008 Wiley‐Liss, Inc.     

Head region of unconventional myosin I family members is responsible for the organ‐specificity of their roles in left–right polarity in Drosophila

In Drosophila, Myosin31DF (Myo31DF), encoding a Myosin ID protein, has crucial roles in left–right (LR) asymmetric development. Loss of Myo31DF function leads to laterality inversion for many organs, including the embryonic gut. Here, we found that Myo31DF was required before LR asymmetric morphogenesis in the hindgut, suggesting it functions in LR patterning instead of directly in hindgut morphological changes. Myosin61F (Myo61F) encodes another Myosin I, and Myo31DF or Myo61F overexpression reverses the laterality of different organs. Myo31DF and Myo61F have domains conserved in Myosin proteins, particularly in the proteins' head regions. We studied the roles of these domains in LR patterning using overexpression analysis. The Actin‐binding and ATP‐binding domains were essential for both proteins, but the IQ domains, binding sites for Myosin light chains, were required only by Myo31DF. Our results also suggest that the organ specificities of the Myo31DF and Myo61F activities depended on their head regions. Developmental Dynamics 237:3528–3537, 2008. © 2008 Wiley‐Liss, Inc.     

Calcium signaling: A common thread in vertebrate left–right axis development

The establishment of a left–right axis during vertebrate development is essential for coordinating the relative positions of the internal organs to ensure that they function appropriately. Studies in numerous model organisms have revealed differences in regulative mechanisms upstream of nodal signaling, a conserved pathway in left–right axis specification. This review will summarize the diverse pathways involved in the break of left–right symmetry and explore in depth the multiple roles of calcium in vertebrate left–right axis specification. Developmental Dynamics 237:3491–3496, 2008. © 2008 Wiley‐Liss, Inc.     

Fluid dynamics of nodal flow and left–right patterning in development

The manner in which the nodal flow determines the breaking of left–right symmetry during development is a beautiful example of the application of fluid dynamics to developmental biology. Detailed understanding of this crucial developmental process has greatly advanced by the transfer of ideas between these two disciplines. In this article, we review our and others' work on applying fluid dynamics and dynamical systems to the problem of left–right symmetry breaking in vertebrates. Developmental Dynamics 237:3477–3490, 2008. © 2008 Wiley‐Liss, Inc.     

Classification of left–right patterning defects in zebrafish,mice, and humans

Numerous genes and developmental processes have been implicated in the establishment of the vertebrate left–right axis. Although the mechanisms that initiate left–right patterning may be distinct in different classes of vertebrates, it is clear that the asymmetric gene expression patterns of nodal, lefty, and pitx2 in the left lateral plate mesoderm are conserved and that left–right development of the brain, heart, and gut is tightly linked to the development of the embryonic midline. This review categorizes left–right patterning defects based on asymmetric gene expression patterns, midline phenotypes, and situs phenotypes. In so doing, we hope to provide a framework to assess the genetic bases of laterality defects in humans and other vertebrates. © 2001 Wiley‐Liss, Inc.     

Sox17 and chordin are required for formation of Kupffer's vesicle and left‐right asymmetry determination in zebrafish

Kupffer's vesicle (KV), a ciliated fluid‐filled sphere in the zebrafish embryo with a critical role in laterality determination, is derived from a group of superficial cells in the organizer region of the gastrula named the dorsal forerunner cells (DFC). We have examined the role of the expression of sox17 and chordin (chd) in the DFC in KV formation and laterality determination. Whereas sox17 was known to be expressed in DFC, its function in these cells was not studied before. Further, expression of chd in these cells has not been reported previously. Targeted knockdown of Sox17 and Chd in DFC led to aberrant Left‐Right (L‐R) asymmetry establishment, as visualized by the expression of southpaw and lefty, and heart and pancreas placement in the embryo. These defects correlated with the formation of small KVs with apparently diminished cilia, consistent with the known requirement for ciliary function in the laterality organ for the establishment of L‐R asymmetry. Developmental Dynamics 239:2980–2988, 2010. Published 2010 Wiley‐Liss, Inc.     

Dissecting the role of Fgf signaling during gastrulation and left‐right axis formation in mouse embryos using chemical inhibitors

Fgf signaling plays pivotal roles in mouse gastrulation and left‐right axis formation. However, although genetic analyses have revealed important aspects of Fgf signaling in these processes, the temporal resolution of genetic studies is low. Here, we combined whole‐embryo culture with application of chemical compounds to inhibit Fgf signaling at specific time points. We found that sodium chlorate and PD173074 are potent inhibitors of Fgf signaling in early mouse embryos. Fgf signaling is required for the epithelial‐to‐mesenchymal transition of the primitive streak before the onset of gastrulation. Once gastrulation begins, Fgf signaling specifies mesodermal fates via the Ras/MAPK downstream cascade. Finally, Fgf signaling on the posterior side of the embryo during gastrulation induces Nodal expression in the node via Tbx6‐Dll1, the initial event required for Nodal expression in the left lateral plate mesoderm. Developmental Dynamics 239:1768–1778, 2010. © 2010 Wiley‐Liss, Inc.     

Cerberus functions as a BMP agonist to synergistically induce nodal expression during left–right axis determination in the chick embryo

Left‐sided expression of Nodal in the lateral plate mesoderm (LPM) during early embryogenesis is a crucial step in establishing the left–right (L–R) axis in vertebrates. In the chick, it was suggested that chick Cerberus (cCer), a Cerberus/Dan family member, induces Nodal expression by antagonizing bone morphogenetic protein (BMP) activity in the left LPM. In contrast, it has also been shown that BMPs positively regulate Nodal expression in the left LPM in the chick embryo. Thus, it is still unclear how the bilaterally expressed BMPs induce Nodal expression only in the left LPM. In this study, we demonstrate that BMP signaling is necessary and sufficient for the induction of Nodal expression in the chick LPM where the type I BMP receptor‐IB (BMPR‐IB) likely mediates this induction. Tissue grafting experiments indicate the existence of a Nodal inductive factor in the left LPM rather than the presence of a Nodal inhibitory factor in the right LPM. We demonstrate that cCer functions as a BMP agonist instead of antagonist, being able to enhance BMP signaling in cell culture. This conclusion is further supported by the immunoprecipitation assays that provide convincing biochemical evidence for a direct interaction between cCer and BMP receptor. Because cCer is expressed restrictedly in the left LPM, BMPs and cCer appear to act synergistically to activate Nodal expression in the left LPM in the chick. Developmental Dynamics 237:3613–3623, 2008. © 2008 Wiley‐Liss, Inc.     

Morphogenesis of the node and notochord: The cellular basis for the establishment and maintenance of left–right asymmetry in the mouse

Establishment of left–right asymmetry in the mouse embryo depends on leftward laminar fluid flow in the node, which initiates a signaling cascade that is confined to the left side of the embryo. Leftward fluid flow depends on two cellular processes: motility of the cilia that generate the flow and morphogenesis of the node, the structure where the cilia reside. Here, we provide an overview of the current understanding and unresolved questions about the regulation of ciliary motility and node structure. Analysis of mouse mutants has shown that the motile cilia must have a specific structure and length, and that they must point posteriorly to generate the necessary leftward fluid flow. However, the precise structure of the motile cilia is not clear and the mechanisms that position cilia on node cells have not been defined. The mouse node is a teardrop‐shaped pit at the distal tip of the early embryo, but the morphogenetic events that create the mature node from cells derived from the primitive streak are only beginning to be characterized. Recent live imaging experiments support earlier scanning electron microscopy (SEM) studies and show that node assembly is a multi‐step process in which clusters of node precursors appear on the embryo surface as overlying endoderm cells are removed. We present additional SEM and confocal microscopy studies that help define the transition stages during node morphogenesis. After the initiation of left‐sided signaling, the notochordal plate, which is contiguous with the node, generates a barrier at the embryonic midline that restricts the cascade of gene expression to the left side of the embryo. The field is now poised to dissect the genetic and cellular mechanisms that create and organize the specialized cells of the node and midline that are essential for left–right asymmetry. Developmental Dynamics 237:3464–3476, 2008. © 2008 Wiley‐Liss, Inc.     

Initiation and propagation of posterior to anterior (PA) waves in zebrafish left–right development

One of the most highly conserved steps in left–right patterning is asymmetric gene expression in lateral plate mesoderm (LPM). Here, we quantitatively describe the timing of the posterior to anterior (PA) wave‐like propagation of zebrafish southpaw (Nodal) and pitx2 in LPM and lefty1 in the midline. By altering the timing of the PA wave, we provide evidence that the PA wave in the LPM instructs brain asymmetry. We find that initiation of pitx2 in LPM and lefty1 in midline depends on Southpaw, and that casanova (sox32) and two Nodal inhibitors, lefty1 and charon, have distinct roles upstream of PA wave initiation. Surprisingly, Casanova, endoderm and Kupffer's Vesicle are not required for normal timing of southpaw initiation and PA propagation. In contrast, lefty1 morphants display precocious asymmetric initiation of southpaw with an intrinsic left‐hand orientation, whereas charon morphants have premature initiation without LR orientation, indicating distinct roles for these Nodal antagonists. Developmental Dynamics 237:3640–3647, 2008. © 2008 Wiley‐Liss, Inc.     

ID2 and ID3 are indispensable for Th1 cell differentiation during influenza virus infection in mice

Antigen‐specific Th1 cells could be a passage to the infection sites during infection to execute effector functions, such as help CD8⁺ T cells to localize in these sites by secretion of anti‐viral cytokines‐IFN‐γ or direct cytotoxicity of antigen‐bearing cells. However, the molecular components that modulate Th1 cell differentiation and function in response to viral infection remain incompletely understood. Here, we reported that both inhibitor of DNA binding 3(Id3) protein and inhibitor of DNA binding 2(Id2) protein promoted Th1 cell differentiation. Depletion of Id3 or Id2 led to severe defect of Th1 cell differentiation during influenza virus infection. Whereas depletion of both Id3 and Id2 in CD4⁺ T cells restrained Th1 cell differentiation to a greater extent, indicating that Id3 and Id2 nonredundantly regulate Th1 cell differentiation. Moreover, deletion of E‐proteins, the antagonists of Id proteins, greatly enhanced Th1 cell differentiation. Mechanistic study indicated that E‐proteins suppressed Th1 cell differentiation by directly binding to the regulatory elements of Th1 cell master regulator T‐bet and regulate T‐bet expression. Thus, our findings identified Id‐protein's importance for Th1 cells and clarified the nonredundant role of Id3 and Id2 in regulating Th1 cell differentiation, providing novel insight that Id3‐Id2‐E protein axis are essential for Th1 cell polarization.     

Man1, an inner nuclear membrane protein,regulates left–right axis formation by controlling nodal signaling in a node‐independent manner

Man1, an inner nuclear membrane protein, regulates transforming growth factor β signaling by interacting with receptor‐associated Smads. In Man1‐deficient (Man1^Δ/Δ) embryos, vascular remodeling is perturbed by misregulation of Smad activity. Here, we show that Man1^Δ/Δ embryos exhibit abnormal heart morphogenesis including the looping abnormality. We searched for the molecular basis underlying the heart abnormalities and found that the left side‐specific genes responsible for left–right (LR) asymmetry, Nodal, Lefty2, and Pitx2, were expressed bilaterally in the lateral plate mesoderm and that their expression was enhanced significantly in mutants. Notably, Lefty1, a marker for the midline barrier, was maintained in Man1^Δ/Δ mutants. Crossing Man1^Δ/+ with Nodal hypomorphs (Nodal^neo/+), in which Nodal signaling in the node is disrupted, to generate double homozygous embryos (Man1^Δ/Δ; Nodal^neo/neo) revealed that the bilateral Nodal was retained in Man1^Δ/Δ; Nodal^neo/neo embryos. These results suggest that Man1 regulates LR asymmetry by controlling Nodal signaling in a node‐independent manner. Developmental Dynamics 237:3565–3576, 2008. © 2008 Wiley‐Liss, Inc.     

Primordial germ cells in the dorsal mesentery of the chicken embryo demonstrate left–right asymmetry and polarized distribution of the EMA1 epitope

Despite the importance of the chicken as a model system, our understanding of the development of chicken primordial germ cells (PGCs) is far from complete. Here we characterized the morphology of PGCs at different developmental stages, their migration pattern in the dorsal mesentery of the chicken embryo, and the distribution of the EMA1 epitope on PGCs. The spatial distribution of PGCs during their migration was characterized by immunofluorescence on whole‐mounted chicken embryos and on paraffin sections, using EMA1 and chicken vasa homolog antibodies. While in the germinal crescent PGCs were rounded and only 25% of them were labeled by EMA1, often seen as a concentrated cluster on the cell surface, following extravasation and migration in the dorsal mesentery PGCs acquired an elongated morphology, and 90% exhibited EMA1 epitope, which was concentrated at the tip of the pseudopodia, at the contact sites between neighboring PGCs. Examination of PGC migration in the dorsal mesentery of Hamburger and Hamilton stage 20–22 embryos demonstrated a left–right asymmetry, as migration of cells toward the genital ridges was usually restricted to the right, rather than the left, side of the mesentery. Moreover, an examination of another group of cells that migrate through the dorsal mesentery, the enteric neural crest cells, revealed a similar preference for the right side of the mesentery, suggesting that the migratory pathway of PGCs is dictated by the mesentery itself. Our findings provide new insights into the migration pathway of PGCs in the dorsal mesentery, and suggest a link between EMA1, PGC migration and cell–cell interactions. These findings may contribute to a better understanding of the mechanism underlying migration of PGCs in avians.     

Morphological and molecular left–right asymmetries in the development of the proepicardium: A comparative analysis on mouse and chick embryos

The proepicardium (PE) is an embryonic progenitor cell population that delivers the epicardium, the majority of the cardiac interstitium, and the coronary vasculature. In the present study, we compared PE development in mouse and chick embryos. In the mouse, a left and a right PE anlage appear simultaneously, which subsequently merge at the embryonic midline to form a single PE. In chick embryos, the right PE anlage appears earlier than the left and only the right anlage acquires the full PE‐phenotype. The left anlage remains in a rudimentary state. The expression patterns of PE marker genes (Tbx18, Wt1) correspond to the morphological data, being bilateral in the mouse and unilateral in the chick. Bmp4, which is unilaterally expressed in the right PE of chick embryos, is symmetrically expressed in the sinus venosus wall cranial to the PE in mouse embryos. Asymmetric development of the chicken PE might reflect side‐specific differences in topographical relationships to tissues with PE‐inducing or repressing activity or might result from the PE‐repressing activity of the right PE, which grows earlier. To test these hypotheses, we analyzed PE development in chick embryos, firstly, subsequent to experimentally induced inversion of PE topographical relationships to neighbouring tissues; secondly, in organ cultures; and, thirdly, subsequent to induction of cardia bifida. In all three experiments, only the right PE develops the full PE phenotype. Our results suggest that PE development might be controlled by the L–R pathway in the chick but not in the mouse embryo. Developmental Dynamics 236:684–695, 2007. © 2007 Wiley‐Liss, Inc.     

Selected signalling proteins recruited to the T‐cell receptor–CD3 complex

The T‐cell receptor (TCR)–CD3 complex, expressed on T cells, determines the outcome of a T‐cell response. It consists of the TCR‐αβ heterodimer and the non‐covalently associated signalling dimers of CD3εγ, CD3εδ and CD3ζζ. TCR‐αβ binds specifically to a cognate peptide antigen bound to an MHC molecule, whereas the CD3 subunits transmit the signal into the cytosol to activate signalling events. Recruitment of proteins to specialized localizations is one mechanism to regulate activation and termination of signalling. In the last 25 years a large number of signalling molecules recruited to the TCR–CD3 complex upon antigen binding to TCR‐αβ have been described. Here, we review knowledge about five of those interaction partners: Lck, ZAP‐70, Nck, WASP and Numb. Some of these proteins have been targeted in the development of immunomodulatory drugs aiming to treat patients with autoimmune diseases and organ transplants.     

Immunohistochemical expression of multidrug resistance proteins in mature T/NK‐cell lymphomas

Multidrug resistance (MDR) is defined as resistance of tumor cells to a wide spectrum of structurally and functionally unrelated drugs. One of the most important mechanisms in mediating MDR is that involving cellular drug efflux transporters. Drug resistance is a common and formidable obstacle to therapy in mature T/NK‐cell lymphomas and the MDR phenotype is thought to be one of the contributing mechanisms. In this study we assessed the immunohistochemical expression of P‐gp (P‐glycoprotein), MRP‐1 (multidrug resistance associated protein 1), BCRP (breast cancer resistance protein) and LRP (lung resistance protein) in 45 mature T/NK‐cell lymphomas diagnosed at our hospital. We detected P‐gp expression in 31% (13/42), MRP‐1 expression in 74% (31/42), BCRP in 78% (32/ 41) and LRP in 59% (26/44) of the cases. These findings show that our T/NK‐cell lymphoma cases display high frequency of MDR protein expression.     

LRIG1 regulates cadherin‐dependent contact inhibition directing epithelial homeostasis and pre‐invasive squamous cell carcinoma development

Epidermal growth factor receptor (EGFR) pathway activation is a frequent event in human carcinomas. Mutations in EGFR itself are, however, rare, and the mechanisms regulating EGFR activation remain elusive. Leucine‐rich immunoglobulin repeats‐1 (LRIG1), an inhibitor of EGFR activity, is one of four genes identified that predict patient survival across solid tumour types including breast, lung, melanoma, glioma, and bladder. We show that deletion of Lrig1 is sufficient to promote murine airway hyperplasia through loss of contact inhibition and that re‐expression of LRIG1 in human lung cancer cells inhibits tumourigenesis. LRIG1 regulation of contact inhibition occurs via ternary complex formation with EGFR and E‐cadherin with downstream modulation of EGFR activity. We find that LRIG1 LOH is frequent across cancers and its loss is an early event in the development of human squamous carcinomas. Our findings imply that the early stages of squamous carcinoma development are driven by a change in amplitude of EGFR signalling governed by the loss of contact inhibition. © 2012 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Prostaglandin E2 and its receptor EP2 trigger signaling that contributes to YAP‐mediated cell competition

Cell competition is a biological process by which unfit cells are eliminated from “cell society.” We previously showed that cultured mammalian epithelial Madin‐Darby canine kidney (MDCK) cells expressing constitutively active YAP were eliminated by apical extrusion when surrounded by “normal” MDCK cells. However, the molecular mechanism underlying the elimination of active YAP‐expressing cells was unknown. Here, we used high‐throughput chemical compound screening to identify cyclooxygenase‐2 (COX‐2) as a key molecule triggering cell competition. Our work shows that COX‐2‐mediated PGE₂ secretion engages its receptor EP2 on abnormal and nearby normal cells. This engagement of EP2 triggers downstream signaling via an adenylyl cyclase‐cyclic AMP‐PKA pathway that, in the presence of active YAP, induces E‐cadherin internalization leading to apical extrusion. Thus, COX‐2‐induced PGE₂ appears a warning signal to both abnormal and surrounding normal cells to drive cell competition.