Cilia propel the embryo in the right direction

Cilia have long been suspected to play a role in the determination of left-right asymmetry. Humans with the dominantly inherited condition Kartagener syndrome have defective cilia and a 50% incidence of mirror-image positioning of their organs (situs inversus). Analysis of mouse mutations affecting ciliary biogenesis and motility has demonstrated that the molecular motors kinesin and dynein are required to establish normal handed organismal asymmetry. The cilia that propel formation of the embryonic left-right axis are not conventional cilia, but monocilia. They are found on the node, or organizer, of the gastrulation-stage mouse embryo where they drive net leftward movement of the fluid surrounding the node, and initiate left-right asymmetry.     

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.     

Targeted mutagenesis in the sea urchin embryo using zinc‐finger nucleases

We showed that engineered zinc‐finger nucleases (ZFNs), which consist of a zinc‐finger DNA‐binding array and a nuclease domain of the restriction enzyme FokI, can introduce mutations at a specific genomic site in the sea urchin embryo. Using bacterial one‐hybrid screening with zinc‐finger randomized libraries and a single‐strand annealing assay in cultured cells, ZFNs targeting the sea urchin Hemicentrotus pulcherrimus homologue of HesC (HpHesC) were efficiently selected. Consistent with the phenotype observed in embryos injected with an antisense morpholino oligonucleotide against HpHesC, an increase in the primary mesenchyme cell population was observed in embryos injected with a pair of HpHesC ZFN mRNAs. In addition, sequence analysis of the mutations showed that deletions and insertions occurred at the HpHesC target site in the embryos injected with the HpHesC ZFN mRNAs. These results suggest that targeted gene disruption using ZFNs is feasible for the sea urchin embryo.     

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.     

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.     

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.     

oleed,a medaka Polycomb group gene,regulates ciliogenesis and left–right patterning

Left‐right (LR) patterning is an essential part of the animal body plan. Primary cilia are known to play a pivotal role in this process. In humans, genetic disorders of ciliogenesis cause serious congenital diseases. A comprehensive mechanism that regulates ciliogenesis has not been proposed so far. Here, we show that EED, a core member of the Polycomb group (PcG) genes and a presumed player in many epigenetic processes, is required for ciliogenesis and subsequent LR patterning in the medaka fish, Oryzias latipes. Moderate knockdown of oleed, a medaka homolog of EED, preferentially caused situs inversus. In the affected embryo, the cilia in Kupffer’s vesicle showed various defects in their structure, position and motility. Furthermore, we demonstrated that oleed maintains the expression of Noto, which, in mice, regulates ciliogenesis and LR patterning. This study provides the first evidence for the involvement of epigenetic plasticity in LR patterning through ciliogenesis.     

Intersecting batteries of differentially expressed genes in the early sea urchin embryo

We determined the distribution of cis-regulatory sites, previously identified in the control domain of the CyIIIa gene, in three other genes displaying diverse spatial patterns of expression in the sea urchin embryo. Competitive gel-shift reactions were carried out using probes from the CyIIIa gene, with competitor fragments isolated from the previously defined control domains of the other genes. CyIIIa is expressed only in aboral ectoderm lineages; the other genes studied were Spec1, also expressed in aboral ectoderm; CyI, expressed in many different cell types; and SM50, expressed only in skeletogenic mesenchyme. All four genes are activated at about the same time in late cleavage. Where competitive interactions indicated a functionally comparable binding site (in vitro), a sequence homology was sought, and in most cases could be identified. An interesting pattern of putative regulatory site usage emerges: Of 10 CyIIIa interactions tested, three only were unique to the CyIIIa gene with respect to the set of four genes tested; one believed on previous evidence to be a temporal regulator was shared by all four genes, and the remainder were shared in various subsets of the four genes.     

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.     

The Ars insulator facilitates I-sceI meganuclease-mediated transgenesis in the sea urchin embryo

For the efficient generation of transgenic sea urchins, we have adopted an I-SceI meganuclease-mediated transgenesis method. Several types of promoter-GFP gene constructs flanked by two I-SceI recognition sequences were co-injected with I-SceI into sea urchin fertilized eggs. Using cell-lineage-specific promoter constructs, the frequency of transgene expression was elevated, and their level of mozaicism was reduced. The addition of the Ars insulator sequence, which is known to block the enhancer activity and protect transgenes from position effects, led to a reduction in ectopic transgene expression and an elevation of transgene expression frequency in this I-SceI-mediated system. However, the magnitude of the effects of the Ars insulator was dependent upon the promoter constructs. QPCR analysis also showed that the Ars insulator increases the transgene copy number. These results suggest that the I-SceI-mediated method using the Ars insulator is advantageous for transgenesis in the sea urchin embryo. Developmental Dynamics 237:2475-2482, 2008. (c) 2008 Wiley-Liss, Inc.     

Lineage and fate of each blastomere of the eight-cell sea urchin embryo

A fluoresceinated lineage tracer was injected into individual blastomeres of eight-cell sea urchin (Strongylocentrotus purpuratus) embryos, and the location of the progeny of each blastomere was determined in the fully developed pluteus. Each blastomere gives rise to a unique portion of the advanced embryo. We confirm many of the classical assignments of cell fate along the animal-vegetal axis of the cleavage-stage embryo, and demonstrate that one blastomere of the animal quartet at the eight-cell stage lies nearest the future oral pole and the opposite one nearest the future aboral pole of the embryo. Clones of cells deriving from ectodermal founder cells always remain contiguous, while clones of cells descendant from the vegetal plate (i.e., gut, secondary mesenchyme) do not. The locations of ectodermal clones contributed by specific blastomeres require that the larval plane of bilateral symmetry lie approximately equidistant (i.e., at a 45 degree angle) from each of the first two cleavage planes. These results underscore the conclusion that many of the early spatial patterns of differential gene expression observed at the molecular level are specified in a clonal manner early in embryonic sea urchin development, and are each confined to cell lineages established during cleavage.     

Influence of cell polarity on early development of the sea urchin embryo

Background: Establishment and maintenance of cell polarity is critical for normal embryonic development. Previously, it was thought that the echinoderm embryo remained relatively unpolarized until the first asymmetric division at the 16‐cell stage. Here, we analyzed roles of the cell polarity regulators, the PAR complex proteins, and how their disruption in early development affects later developmental milestones. Results: We found that PAR6, aPKC, and CDC42 localize to the apical cortex as early as the 2‐cell stage and that this localization is retained through the gastrula stage. Of interest, PAR1 also colocalizes with these apical markers through the gastrula stage. Additionally, PAR1 was found to be in complex with aPKC, but not PAR6. PAR6, aPKC, and CDC42 are anchored in the cortical actin cytoskeleton by assembled myosin. Furthermore, assembled myosin was found to be necessary to maintain proper PAR6 localization through subsequent cleavage divisions. Interference with myosin assembly prevented the embryos from reaching the blastula stage, while transient disruptions of either actin or microtubules did not have this effect. Conclusions: These observations suggest that disruptions of the polarity in the early embryo can have a significant impact on the ability of the embryo to reach later critical stages in development. Developmental Dynamics 244:1469–1484, 2015. © 2015 Wiley Periodicals, Inc.     

E and ID proteins regulate cell chirality and left–right asymmetric development in Drosophila

How left–right (LR) asymmetric forms in the animal body is a fundamental problem in Developmental Biology. Although the mechanisms for LR asymmetry are well studied in some species, they are still poorly understood in invertebrates. We previously showed that the intrinsic LR asymmetry of cells (designated as cell chirality) drives LR asymmetric development in the Drosophila embryonic hindgut, although the machinery of the cell chirality formation remains elusive. Here, we found that the Drosophila homologue of the Id gene, extra macrochaetae (emc), is required for the normal LR asymmetric morphogenesis of this organ. Id proteins, including Emc, are known to interact with and inhibit E‐box‐binding proteins (E proteins), such as Drosophila Daughterless (Da). We found that the suppression of da by wild‐type emc was essential for cell chirality formation and for normal LR asymmetric development of the embryonic hindgut. Myosin ID (MyoID), which encodes the Drosophila Myosin ID protein, is known to regulate cell chirality. We further showed that Emc‐Da regulates cell chirality formation, in which Emc functions upstream of or parallel to MyoID. Abnormal Id‐E protein regulation is involved in various human diseases. Our results suggest that defects in cell shape may contribute to the pathogenesis of such diseases.     

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.     

Nodal/activin signaling establishes oral-aboral polarity in the early sea urchin embryo.

Components of the Wnt signaling pathway are involved in patterning the sea urchin primary or animal-vegetal (AV) axis, but the molecular cues that pattern the secondary embryonic axis, the aboral/oral (AO) axis, are not known. In an analysis of signaling molecules that influence patterning along the sea urchin embryonic axes, we found that members of the activin subfamily of transforming growth factor-beta (TGF-beta) signaling molecules influence the establishment of AO polarities in the early embryo. Injection of activin mRNAs into fertilized eggs or treatment with exogenously applied recombinant activin altered the allocation of ectodermal fates and ventralized the embryo. The phenotypes observed resemble the ventralized phenotype previously reported for NiCl2, a known disrupter of AO patterning. Sensitivity to exogenous activin occurs between fertilization and the late blastula stage, which is also the time of highest NiCl2 sensitivity. These results argue that specification of fates along the embryonic AO axis involves TGF-beta signaling. To further examine TGF-beta signaling in these embryos, we cloned an endogenous TGF-beta from sea urchin embryos that is a member of the activin subfamily, SpNodal, and show through gain of function analysis that it recapitulates results obtained with exogenous activins and NiCl2. The expression pattern of SpNodal is consistent with a role for nodal signaling in the establishment of fates along the AO axis. Loss of function experiments using SpNodal antisense morpholinos also support a role for SpNodal in the establishment of the AO axis.     

A putative role for carbohydrates in sea urchin gastrulation.

Many studies have examined the effects of lectins on embryonic development. Recently, it has been shown that lectins actually enter the blastocoel of sea urchin embryos without microinjection and bind to specific cell types. The present study was performed to examine the effects of lectins on sea urchin gastrulation. Strongylocentrotus purpuratus sea urchin embryos were incubated with several lectins at concentrations from 0.01 microgram/ml to 100 micrograms/ml at 15-28 h in the presence or absence of the preferential binding sugars. The most interesting findings were that the mannose specific lectins Lens culinaris agglutinin (LcH) which binds to secondary mesenchyme cells involved in archenteron anchoring and Pisum sativum (PSA) caused exogastrulation. Wheat germ agglutinin (WGA) which binds to primary mesenchyme cells involved in skeletogenesis caused defective skeletogenesis. Our findings suggest that D-mannose-like residues (LcH and PSA specific sugar) may function in archenteron development and anchoring, while N-acetyl-D-glucosamine-like groups (WGA specific sugar) may contribute to control of primary mesenchyme positioning and function. Specific carbohydrate-containing receptors may, therefore, be of importance in specific gastrulation events.