Left-right patterning in the mouse requires Epb4.1l5-dependent morphogenesis of the node and midline

The mouse node is a transient early embryonic structure that is required for left-right asymmetry and for generation of the axial midline, which patterns neural and mesodermal tissues. The node is a shallow teardrop-shaped pit that sits at the distal tip of the early headfold (e7.75) embryo. The shape of the node is believed to be important for generation of the coherent leftward fluid flow required for initiation of left-right asymmetry, but little is known about the morphogenesis of the node. Here we show that the FERM domain protein Lulu/Epb4.1l5 is required for left-right asymmetry in the early mouse embryo. Unlike other genes previously shown to be required for left-right asymmetry in the mouse, lulu is not required for specification of node cell identity, for Nodal signaling in the node or for ciliogenesis. Instead, lulu is required for proper morphogenesis of the node and midline. The precursors of the wild-type node undergo a series of rapid morphological transitions. First, node precursors arise from an epithelial-to-mesenchymal transition at the anterior primitive streak. While in the mesenchymal layer, the node precursors form several ciliated rosette-like clusters; they then rapidly undergo a mesenchymal-to-epithelial transition to insert into the outer, endodermal layer of the embryo. In lulu mutants, node precursor cells are specified and form clusters, but those clusters fail to coalesce to make a single continuous node epithelium. The data suggest that the assembly of the contiguous node epithelium from mesenchymal clusters requires a rapid reorganization of apical-basal polarity that depends on Lulu/Epb4.1l5.     

Foxj1 regulates asymmetric gene expression during left-right axis patterning in mice

Mice with a targeted mutation of the foxj1 gene demonstrate either D- or L-looping of the embryonic cardiac tube. Foxj1 is expressed in ventral cells of the embryonic node prior to asymmetric, left-right expression of other genes. Despite an absence of 9+2 cilia in foxj1(-/-) mice, 9+0 cilia are present in the node of foxj1(-/-) embryos. In foxj1(-/-) embryos, the patterns of expression of the TGF-beta family member nodal and the homeobox family member pitx2 are randomized. No expression of the TGF-beta family member lefty-2 is observed in any foxj1(-/-) early somite stage embryos. Foxj1 thus acts early in left-right axis patterning and regulates asymmetric gene expression. This regulation does not appear to be the result of a direct interaction between Foxj1 and the genes examined.     

Breakthroughs and future challenges in left–right patterning

How left–right (LR) asymmetry emerges during development has been a classic problem in the field of developmental biology, but it is only since the 1990s that molecular and genetic approaches have become possible. Since current progress in LR patterning has been summarized in several other reviews, I would like to briefly describe how this topic developed in the early days and what remains to be solved. In particular, two breakthroughs in the mid to late 1990s made major contributions to recent progress. Despite the rapid progress, there remain many challenging issues that need to be addressed in future.     

Left-right asymmetry in embryonic development: a comprehensive review

Embryonic morphogenesis occurs along three orthogonal axes. While the patterning of the anterior-posterior and dorsal-ventral axes has been increasingly well characterized, the left-right (LR) axis has only recently begun to be understood at the molecular level. The mechanisms which ensure invariant LR asymmetry of the heart, viscera, and brain represent a thread connecting biomolecular chirality to human cognition, along the way involving fundamental aspects of cell biology, biophysics, and evolutionary biology. An understanding of LR asymmetry is important not only for basic science, but also for the biomedicine of a wide range of birth defects and human genetic syndromes. This review summarizes the current knowledge regarding LR patterning in a number of vertebrate and invertebrate species, discusses several poorly understood but important phenomena, and highlights some important open questions about the evolutionary origin and conservation of mechanisms underlying embryonic asymmetry.     

BMP signaling through ACVRI is required for left-right patterning in the early mouse embryo

Vertebrate organisms are characterized by dorsal-ventral and left-right asymmetry. The process that establishes left-right asymmetry during vertebrate development involves bone morphogenetic protein (BMP)-dependent signaling, but the molecular details of this signaling pathway remain poorly defined. This study tests the role of the BMP type I receptor ACVRI in establishing left-right asymmetry in chimeric mouse embryos. Mouse embryonic stem (ES) cells with a homozygous deletion at Acvr1 were used to generate chimeric embryos. Chimeric embryos were rescued from the gastrulation defect of Acvr1 null embryos but exhibited abnormal heart looping and embryonic turning. High mutant contribution chimeras expressed left-side markers such as nodal bilaterally in the lateral plate mesoderm (LPM), indicating that loss of ACVRI signaling leads to left isomerism. Expression of lefty1 was absent in the midline of chimeric embryos, but shh, a midline marker, was expressed normally, suggesting that, despite formation of midline, its barrier function was abolished. High-contribution chimeras also lacked asymmetric expression of nodal in the node. These data suggest that ACVRI signaling negatively regulates left-side determinants such as nodal and positively regulates lefty1. These functions maintain the midline, restrict expression of left-side markers, and are required for left-right pattern formation during embryogenesis in the mouse.     

The roles of Groucho/Tle in left-right asymmetry and Kupffer's vesicle organogenesis

The heart is the first organ to form and function in the vertebrate embryo. Furthermore, differences between the left and right sides of the embryo become first detectable during cardiac development. We observed strong cardiac laterality phenotypes in medaka embryos by manipulating Groucho protein activity. The phenotypes produced by misexpressing Tle4 and the dominant-negative Aes reveal a general effect of these corepressor proteins on left-right (LR) development. With the help of an inducible expression system, we were able to define temporally different phases for these effects. In an early phase during gastrulation, Groucho proteins regulate Brachyury expression in the dorsal forerunner cells, which later gives rise to the Kupffer's vesicle (KV). The interference of endogenous Groucho proteins by misexpression of Aes leads to KVs of reduced size, whereas overexpression of Tle4 results in enlarged KVs. The expression level of the cilia marker Lrd was also affected both positively and negatively from these treatments. In the late phase during somitogenesis, Groucho proteins regulate the asymmetric activities of Nodal and Lefty genes. Altering canonical Wnt signaling produced similar results in late embryos, however, this did not affect KV morphogenesis or Lrd expression in early embryos. Therefore, changes in Kupffer's vesicle morphogenesis and the laterality of visceral organs following alterations in Groucho corepressor levels demonstrate two distinct phases in which Groucho proteins help establish LR asymmetry in medaka fish.     

Left-right asymmetry and congenital cardiac defects: getting to the heart of the matter in vertebrate left-right axis determination

Cellular and molecular left-right differences that are present in the mesodermal heart fields suggest that the heart is lateralized from its inception. Left-right asymmetry persists as the heart fields coalesce to form the primary heart tube, and overt, morphological asymmetry first becomes evident when the heart tube undergoes looping morphogenesis. Thereafter, chamber formation, differentiation of the inflow and outflow tracts, and position of the heart relative to the midline are additional features of heart development that exhibit left-right differences. Observations made in human clinical studies and in animal models of laterality disease suggest that all of these features of cardiac development are influenced by the embryonic left-right body axis. When errors in left-right axis determination happen, they almost always are associated with complex congenital heart malformations. The purpose of this review is to highlight what is presently known about cardiac development and upstream processes of left-right axis determination, and to consider how perturbation of the left-right body plan might ultimately result in particular types of congenital heart defects.     

Dynein axonemal intermediate chain 2 is required for formation of the left-right body axis and kidney in medaka

Ciliary defects lead to various diseases, such as primary ciliary dyskinesia (PCD) and polycystic kidney disease (PKD). We isolated a medaka mutant mii, which exhibits defects in the left-right (LR) polarity of organs, and found that mii encodes dynein axonemal intermediate chain 2a (dnai2a). Ortholog mutations were recently reported to cause PCD in humans. mii mutant embryos exhibited loss of nodal flow in Kupffer's Vesicle (KV), which is equivalent to the mammalian node, and abnormal expression of the left-specific gene. KV cilia in the mii mutant were defective in their outer dynein arms (ODAs), indicating that Dnai2a is required for ODA formation in KV cilia. While the mii mutant retained motility of the renal cilia and failed to show PKD, the loss of dnai2a and another dnai2 ortholog dnai2b led to PKD. These findings demonstrate that Dnai2 proteins control LR polarity and kidney formation through regulation of ciliary motility.     

Sp-Smad2/3 mediates patterning of neurogenic ectoderm by nodal in the sea urchin embryo

Nodal functions in axis and tissue specification during embryogenesis. In sea urchin embryos, Nodal is crucial for specification of oral ectoderm and is thought to pattern neurogenesis in the animal plate. To determine if Nodal functions directly in suppressing neuron differentiation we have prepared mutant forms of Sp-Smad2/3. Expressing an activated form produces embryos similar to embryos overexpressing Nodal, but with fewer neurons. In chimeras in which Nodal is suppressed, cells expressing activated Sp-Smad2/3 form oral ectoderm, but not neurons. In embryos with vegetal signaling blocked, neurons do not form if activated Smad2/3 is co-expressed. Expression of dominant negative mutants produces embryos identical to those resulting from blocking Nodal expression. In chimeras overexpressing Nodal, cells expressing dominant negative Sp-Smad2/3 form aboral ectoderm and give rise to neurons. In permanent blastula chimeras dominant negative Sp-Smad2/3 is able to suppress the effects of Nodal permitting neuron differentiation. In these chimeras Nodal expression in one half suppresses neural differentiation across the interface. Anti-phospho-Smad3 reveals that the cells adjacent to cells expressing Nodal have nuclear immunoreactivity. We conclude Sp-Smad2/3 is a component of the Nodal signaling pathway in sea urchins and that Nodal diffuses short distances to suppress neural differentiation.     

Genetic dissection of nodal function in patterning the mouse embryo

Loss-of-function analysis has shown that the transforming growth factor-like signaling molecule nodal is essential for mouse mesoderm development. However, definitive proof of nodal function in other developmental processes in the mouse embryo has been lacking because the null mutation blocks gastrulation. We describe the generation and analysis of a hypomorphic nodal allele. Mouse embryos heterozygous for the hypomorphic allele and a null allele undergo gastrulation but then display abnormalities that fall into three distinct mutant phenotypic classes, which may result from expression levels falling below critical thresholds in one or more domains of nodal expression. Our analysis of each of these classes provides conclusive evidence for nodal-mediated regulation of several developmental processes in the mouse embryo, beyond its role in mesoderm formation. We find that nodal signaling is required for correct positioning of the anteroposterior axis, normal anterior and midline patterning, and the left-right asymmetric development of the heart, vasculature, lungs and stomach.     

Anterior neural plate regionalization in cripto null mutant mouse embryos in the absence of node and primitive streak

The relation between the role of the organizer at the gastrula stage and the activity of earlier signals in the specification, maintenance, and regionalization of the developing brain anlage is still controversial. Mouse embryos homozygous for null mutation in the cripto gene die at about 9.0 days postcoitum (d.p.c.) and fail to gastrulate and to form the node (the primary organizer). Here, we study the presence and the distribution of anterior neural plate molecular domains in cripto null mutants. We demonstrate that, in cripto(-/-) embryos, the main prosencephalic and mesencephalic regions are present and that they assume the correct topological organization. The identity of the anterior neural domains is maintained in mutant embryos at 8.5 d.p.c., as well as in mutant explants dissected at 8.5 d.p.c. and cultured in vitro for 24 h. Our data imply the existence of a stable neural regionalization of anterior character inside the cripto(-/-) embryos, despite the failure in both the gastrulation process and node formation. These results suggest that, in mouse embryos, the specification of the anterior neural identities can be maintained without an absolute requirement for the embryonic mesoderm and the node.     

Essential roles of fibronectin in the development of the left-right embryonic body plan

Studies in Xenopus laevis suggested that cell-extracellular matrix (ECM) interactions regulate the development of the left–right axis of asymmetry; however, the identities of ECM components and their receptors important for this process have remained unknown. We discovered that FN is required for the establishment of the asymmetric gene expression pattern in early mouse embryos by regulating morphogenesis of the node, while cellular fates of the nodal cells, canonical Wnt and Shh signaling within the node were not perturbed by the absence of FN. FN is also required for the expression of Lefty 1/2 and activation of SMADs 2 and 3 at the floor plate, while cell fate specification of the notochord and the floor plate, as well as signaling within and between these two embryonic organizing centers remained intact in FN-null mutants. Furthermore, our experiments indicate that a major cell surface receptor for FN, integrin α5β1, is also required for the development of the left–right asymmetry, and that this requirement is evolutionarily conserved in fish and mice. Taken together, our studies demonstrate the requisite role for a structural ECM protein and its integrin receptor in the development of the left–right axis of asymmetry in vertebrates.     

Left-right asymmetric morphogenesis of the anterior midgut depends on the activation of a non-muscle myosin II in Drosophila

Many animals exhibit stereotypical left-right (LR) asymmetry in their internal organs. The mechanisms of LR axis formation required for the subsequent LR asymmetric development are well understood, especially in some vertebrates. However, the molecular mechanisms underlying LR asymmetric morphogenesis, particularly how mechanical force is integrated into the LR asymmetric morphogenesis of organs, are poorly understood. Here, we identified zipper (zip), encoding a Drosophila non-muscle myosin II (myosin II) heavy chain, as a gene required for LR asymmetric development of the embryonic anterior midgut (AMG). Myosin II is known to directly generate mechanical force in various types of cells during morphogenesis and cell migration. We found that myosin II was involved in two events in the LR asymmetric development of the AMG. First, it introduced an LR bias to the directional position of circular visceral muscle (CVMU) cells, which externally cover the midgut epithelium. Second, it was required for the LR-biased rotation of the AMG. Our results suggest that myosin II in CVMU cells plays a crucial role in generating the force leading to LR asymmetric morphogenesis. Taken together with previous studies in vertebrates, the involvement of myosin II in LR asymmetric morphogenesis might be conserved evolutionarily.     

Asymmetric Nodal expression in the mouse is governed by the combinatorial activities of two distinct regulatory elements

In all vertebrates, invariant left/right (L/R) positioning and organization of the internal viscera is controlled by a conserved pathway. Nodal, a member of the TGFbeta superfamily is a critical upstream component responsible for initiating L/R axis determination. Asymmetric Nodal expression in the node preceeds and foreshadows morphological L/R asymmetry. Here we address the mechanism of Nodal activation in the left LPM by studying the function of a novel enhancer element, the AIE. We show this element is exclusively active in cells of the left lateral plate mesoderm (LPM) and is not itself responding to Nodal asymmetry. To test the hypothesis that this element may initiate asymmetric Nodal expression in the LPM, we deleted it from the mouse germ line. Mice homozygous for the AIE deletion (Nodal(deltaaie/deltaaie)) show no defects. However, we find that the AIE contributes to regulating the level of asymmetric Nodal activity; analysis of transheterozygous embryos (Nodal(deltaaie/null)) shows reduced Nodal expression in the left LPM associated with a low penetrance of L/R defects. Our findings point to the existence of two independent pathways that control Nodal expression in the left LPM.     

Intraflagellar transport protein 172 is essential for primary cilia formation and plays a vital role in patterning the mammalian brain

IFT172, also known as Selective Lim-domain Binding protein (SLB), is a component of the intraflagellar transport (IFT) complex. In order to evaluate the biological role of the Ift172 gene, we generated a loss-of-function mutation in the mouse. The resulting Slb mutant embryos die between E12.5 and 13.0, and exhibit severe cranio-facial malformations, failure to close the cranial neural tube, holoprosencephaly, heart edema and extensive hemorrhages. Cilia outgrowth in cells of the neuroepithelium is initiated but the axonemes are severely truncated and do not contain visible microtubules. Morphological and molecular analyses revealed a global brain-patterning defect along the dorsal-ventral (DV) and anterior-posterior (AP) axes. We demonstrate that Ift172 gene function is required for early regulation of Fgf8 at the midbrain-hindbrain boundary and maintenance of the isthmic organizer. In addition, Ift172 is required for proper function of the embryonic node, the early embryonic organizer and for formation of the head organizing center (the anterior mesendoderm, or AME). We propose a model suggesting that forebrain and mid-hindbrain growth and AP patterning depends on the early function of Ift172 at gastrulation. Our data suggest that the formation and function of the node and AME in the mouse embryo relies on an indispensable role of Ift172 in cilia morphogenesis and cilia-mediated signaling.