Cell lineage-guided mass spectrometry reveals increased energy metabolism and reactive oxygen species in the vertebrate organizer.

Molecular understanding of the vertebrate Organizer, a tissue center critical for inductive signaling during gastrulation, has so far been mostly limited to transcripts and a few proteins, the latter due to limitations in detection and sensitivity. The Spemann-Mangold Organizer (SMO) in the South African Clawed Frog (X. laevis), a popular model of development, has long been known to be the origin of signals that pattern the mesoderm and central nervous system. Molecular screens of the SMO have identified several genes responsible for the ability of the SMO to establish the body axis. Nonetheless, a comprehensive study of proteins and metabolites produced specifically in the SMO and their functional roles has been lacking. Here, we pioneer a deep discovery proteomic and targeted metabolomic screen of the SMO in comparison to the remainder of the embryo using high-resolution mass spectrometry (HRMS). Quantification of ~4,600 proteins and a panel of targeted metabolites documented differential expression for 460 proteins and multiple intermediates of energy metabolism in the SMO. Upregulation of oxidative phosphorylation and redox regulatory proteins gave rise to elevated oxidative stress and an accumulation of reactive oxygen species in the SMO. Imaging experiments corroborated these findings, discovering enrichment of hydrogen peroxide in the SMO. Chemical perturbation of the redox gradient perturbed mesoderm involution during early gastrulation. HRMS expands the bioanalytical toolbox of cell and developmental biology, providing previously unavailable information on molecular classes to challenge and refine our classical understanding of the Organizer and its function during early patterning of the embryo.

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.

Chiral Cilia Orientation in the Left-Right Organizer.

Chirality is a property of asymmetry between an object and its mirror image. Most biomolecules and many cell types are chiral. In the left-right organizer (LRO), cilia-driven flows transfer such chirality to the body scale. However, the existence of cellular chirality within tissues remains unknown. Here, we investigate this question in Kupffer's vesicle (KV), the zebrafish LRO. Quantitative live imaging reveals that cilia populating the KV display asymmetric orientation between the right and left sides, resulting in a chiral structure, which is different from the chiral cilia rotation. This KV chirality establishment is dynamic and depends on planar cell polarity. While its impact on left-right (LR) symmetry breaking remains unclear, we show that this asymmetry does not depend on the LR signaling pathway or flow. This work identifies a different type of tissue asymmetry and sheds light on chirality genesis in developing tissues.

Mechanical Strain Determines Cilia Length, Motility, and Planar Position in the Left-Right Organizer.

The Xenopus left-right organizer (LRO) breaks symmetry along the left-right axis of the early embryo by producing and sensing directed ciliary flow as a patterning cue. To carry out this process, the LRO contains different ciliated cell types that vary in cilia length, whether they are motile or sensory, and how they position their cilia along the anterior-posterior (A-P) planar axis. Here, we show that these different cilia features are specified in the prospective LRO during gastrulation, based on anisotropic mechanical strain that is oriented along the A-P axis, and graded in levels along the medial-lateral axis. Strain instructs ciliated cell differentiation by acting on a mesodermal prepattern present at blastula stages, involving foxj1. We propose that differential strain is a graded, developmental cue, linking the establishment of an A-P planar axis to cilia length, motility, and planar location during formation of the Xenopus LRO.

On the nature and function of organizers.

Organizers, which comprise groups of cells with the ability to instruct adjacent cells into specific states, represent a key principle in developmental biology. The concept was first introduced by Spemann and Mangold, who showed that there is a cellular population in the newt embryo that elicits the development of a secondary axis from adjacent cells. Similar experiments in chicken and rabbit embryos subsequently revealed groups of cells with similar instructive potential. In birds and mammals, organizer activity is often associated with a structure known as the node, which has thus been considered a functional homologue of Spemann's organizer. Here, we take an in-depth look at the structure and function of organizers across species and note that, whereas the amphibian organizer is a contingent collection of elements, each performing a specific function, the elements of organizers in other species are dispersed in time and space. This observation urges us to reconsider the universality and meaning of the organizer concept.

ADMP controls the size of Spemann's organizer through a network of self-regulating expansion-restriction signals.

BACKGROUND: The bone morphogenetic protein (BMP) signaling gradient is central for dorsoventral patterning in amphibian embryos. This gradient is established through the interaction of several BMPs and BMP antagonists and modulators, some secreted by Spemann's organizer, a cluster of cells coordinating embryonic development. Anti-dorsalizing morphogenetic protein (ADMP), a BMP-like transforming growth factor beta ligand, negatively affects the formation of the organizer, although it is robustly expressed within the organizer itself. Previously, we proposed that this apparent discrepancy may be important for the ability of ADMP to scale the BMP gradient with embryo size, but how this is achieved is unclear. RESULTS: Here we report that ADMP acts in the establishment of the organizer via temporally and mechanistically distinct signals. At the onset of gastrulation, ADMP is required to establish normal organizer-specific gene expression domains, thus displaying a dorsal, organizer-promoting function. The organizer-restricting, BMP-like function of ADMP becomes apparent slightly later, from mid-gastrula. The organizer-promoting signal of ADMP is mediated by the activin A type I receptor, ACVR1 (also known as activin receptor-like kinase-2, ALK2). ALK2 is expressed in the organizer and is required for organizer establishment. The anti-organizer function of ADMP is mediated by ACVRL1 (ALK1), a putative ADMP receptor expressed in the lateral regions flanking the organizer that blocks expansion of the organizer. Truncated ALK1 prevents the organizer-restricting effects of ADMP overexpression, suggesting a ligand-receptor interaction. We also present a mathematical model of the regulatory network controlling the size of the organizer. CONCLUSIONS: We show that the opposed, organizer-promoting and organizer-restricting roles of ADMP are mediated by different receptors. A self-regulating network is proposed in which ADMP functions early through ALK2 to expand its own expression domain, the organizer, and later functions through ALK1 to restrict this domain. These effects are dependent on ADMP concentration, timing, and the spatial localization of the two receptors. This self-regulating temporal switch may control the size of the organizer and the genes expressed within in response to genetic and external stimuli during gastrulation.

Spemann organizer transcriptome induction by early beta-catenin, Wnt, Nodal, and Siamois signals in Xenopus laevis.

The earliest event in Xenopus development is the dorsal accumulation of nuclear ß-catenin under the influence of cytoplasmic determinants displaced by fertilization. In this study, a genome-wide approach was used to examine transcription of the 43,673 genes annotated in the Xenopus laevis genome under a variety of conditions that inhibit or promote formation of the Spemann organizer signaling center. Loss of function of ß-catenin with antisense morpholinos reproducibly reduced the expression of 247 mRNAs at gastrula stage. Interestingly, only 123 ß-catenin targets were enriched on the dorsal side and defined an early dorsal ß-catenin gene signature. These genes included several previously unrecognized Spemann organizer components. Surprisingly, only 3 of these 123 genes overlapped with the late Wnt signature recently defined by two other groups using inhibition by Dkk1 mRNA or Wnt8 morpholinos, which indicates that the effects of ß-catenin/Wnt signaling in early development are exquisitely regulated by stage-dependent mechanisms. We analyzed transcriptome responses to a number of treatments in a total of 46 RNA-seq libraries. These treatments included, in addition to ß-catenin depletion, regenerating dorsal and ventral half-embryos, lithium chloride treatment, and the overexpression of Wnt8, Siamois, and Cerberus mRNAs. Only some of the early dorsal ß-catenin signature genes were activated at blastula whereas others required the induction of endomesoderm, as indicated by their inhibition by Cerberus overexpression. These comprehensive data provide a rich resource for analyzing how the dorsal and ventral regions of the embryo communicate with each other in a self-organizing vertebrate model embryo.

An organizing function of basement membranes in the developing nervous system.

The basement membranes (BMs) of the nervous system include (a) the pial BM that surrounds the entire CNS, (b) the BMs that outline the vascular system of the CNS and PNS and (c) the BMs that are associated with Schwann cells. We previously found that isolated BMs are bi-functionally organized, whereby the two surfaces have different compositional, biomechanical and cell adhesion properties. To find out whether the bi-functional nature of BMs has an instructive function in organizing the tissue architecture of the developing nervous system, segments of human BMs were inserted into (a) the parasomitic mesoderm of chick embryos, intersecting with the pathways of axons and neural crest cells, or (b) into the midline of the embryonic chick spinal cord. The implanted BMs integrated into the embryonic tissues within 24h and were impenetrable to growing axons and migrating neural crests cells. Host axons and neural crest cells contacted the epithelial side but avoided the stromal side of the implanted BM. When the BMs were inserted into the spinal cord, neurons, glia cells and axons assembled at the epithelial side of the implanted BMs, while a connective tissue layer formed at the stromal side, resembling the tissue architecture of the spinal cord at the pial surface. Since the spinal cord is a-vascular at the time of BM implantation, we propose that the bi-functional nature of BMs has the function of segregating epithelial and connective cells into two adjacent compartments and participates in establishing the tissue architecture at the pial surface of the CNS.

Symmetry breakage in the vertebrate embryo: when does it happen and how does it work?

Asymmetric development of the vertebrate embryo has fascinated embryologists for over a century. Much has been learned since the asymmetric Nodal signaling cascade in the left lateral plate mesoderm was detected, and began to be unraveled over the past decade or two. When and how symmetry is initially broken, however, has remained a matter of debate. Two essentially mutually exclusive models prevail. Cilia-driven leftward flow of extracellular fluids occurs in mammalian, fish and amphibian embryos. A great deal of experimental evidence indicates that this flow is indeed required for symmetry breaking. An alternative model has argued, however, that flow simply acts as an amplification step for early asymmetric cues generated by ion flux during the first cleavage divisions. In this review we critically evaluate the experimental basis of both models. Although a number of open questions persist, the available evidence is best compatible with flow-based symmetry breakage as the archetypical mode of symmetry breakage.

Establishment of left-right asymmetry in vertebrate development: the node in mouse embryos.

Establishment of vertebrate left-right asymmetry is a critical process for normal embryonic development. After the discovery of genes expressed asymmetrically along the left-right axis in chick embryos in the mid 1990s, the molecular mechanisms responsible for left-right patterning in vertebrate embryos have been studied extensively. In this review article, we discuss the mechanisms by which the initial symmetry along the left-right axis is broken in the mouse embryo. We focus on the role of primary cilia and molecular mechanisms of ciliogenesis at the node when symmetry is broken and left-right asymmetry is established. The node is considered a signaling center for early mouse embryonic development, and the results we review here have led to a better understanding of how the node functions and establishes left-right asymmetry.

Cilia at the node of mouse embryos sense fluid flow for left-right determination via Pkd2.

Unidirectional fluid flow plays an essential role in the breaking of left-right (L-R) symmetry in mouse embryos, but it has remained unclear how the flow is sensed by the embryo. We report that the Ca(2+) channel Polycystin-2 (Pkd2) is required specifically in the perinodal crown cells for sensing the nodal flow. Examination of mutant forms of Pkd2 shows that the ciliary localization of Pkd2 is essential for correct L-R patterning. Whereas Kif3a mutant embryos, which lack all cilia, failed to respond to an artificial flow, restoration of primary cilia in crown cells rescued the response to the flow. Our results thus suggest that nodal flow is sensed in a manner dependent on Pkd2 by the cilia of crown cells located at the edge of the node.

The head organizer in Hydra.

Organizers and organizing centers play critical roles in axis formation and patterning during the early stages of embryogenesis in many bilaterians. The presence and activity of an organizer was first described in adult Hydra about 100 years ago, and in the following decades organizer regions were identified in a number of bilaterian embryos. In an adult Hydra, the cells of the body column are constantly in the mitotic cycle resulting in continuous displacement of the tissue to the extremities where it is sloughed. In this context, the head organizer located in the hypostome is continuously active sending out signals to maintain the structure and morphology of the head, body column and foot of the animal. The molecular basis of the head organizer involves the canonical Wnt pathway, which acts in a self-renewing manner to maintain itself in the context of the tissue dynamics of Hydra. During bud formation, Hydra's mode of asexual reproduction, a head organizer based on the canonical Wnt pathway is set up to initiate and control the development of a new Hydra. As this pathway plays a central role in vertebrate embryonic organizers, its presence and activity in Hydra indicate that the molecular basis of the organizer arose early in metazoan evolution.

The role of organizers in patterning the nervous system.

The foundation for the anatomical and functional complexity of the vertebrate central nervous system is laid during embryogenesis. After Spemann's organizer and its derivatives have endowed the neural plate with a coarse pattern along its anteroposterior and mediolateral axes, this basis is progressively refined by the activity of secondary organizers within the neuroepithelium that function by releasing diffusible signaling factors. Dorsoventral patterning is mediated by two organizer regions that extend along the dorsal and ventral midlines of the entire neuraxis, whereas anteroposterior patterning is controlled by several discrete organizers. Here we review how these secondary organizers are established and how they exert their signaling functions. Organizer signals come from a surprisingly limited set of signaling factor families, indicating that the competence of target cells to respond to those signals plays an important part in neural patterning.

Two rotating cilia in the node cavity are sufficient to break left-right symmetry in the mouse embryo.

Determination of left-right asymmetry in mouse embryos is achieved by a leftward fluid flow (nodal flow) in the node cavity that is generated by clockwise rotational movement of 200-300 cilia in the node. The precise action of nodal flow and how much flow input is required for the robust read-out of left-right determination remains unknown. Here we show that a local leftward flow generated by as few as two rotating cilia is sufficient to break left-right symmetry. Quantitative analysis of fluid flow and ciliary rotation in the node of mouse embryos shows that left-right asymmetry is already established within a few hours after the onset of rotation by a subset of nodal cilia. Examination of various ciliary mutant mice shows that two rotating cilia are sufficient to initiate left-right asymmetric gene expression. Our results suggest the existence of a highly sensitive system in the node that is able to sense an extremely weak unidirectional flow, and may favour a model in which the flow is sensed as a mechanical force.

Cell cycle arrest in node cells governs ciliogenesis at the node to break left-right symmetry.

Cilia at the node generate a leftward fluid flow that breaks left-right symmetry. However, the molecular mechanisms that regulate ciliogenesis at the node are largely unknown. Here, we show that the epiblast-specific deletion of the gene encoding the BMP type 1 receptor (Acvr1) compromised development of nodal cilia, which results in defects in leftward fluid flow and, thus, abnormalities in left-right patterning. Acvr1 deficiency in mouse embryonic fibroblasts (MEFs) resulted in severe defects in their quiescence-induced primary cilia. Although the induction of quiescence in wild-type MEFs leads to an increase in the level of the cyclin-dependent kinase inhibitor p27(Kip1) and to rapid p27(Kip1) phosphorylation on Ser(10), MEFs deficient in Acvr1 show a reduction in both p27(Kip1) protein levels and in p27(Kip1) Ser(10) phosphorylation. The observed defects in cilium development were rescued by the introduction of p27(Kip1) into Acvr1-deficient MEFs, implying that BMP signaling positively controls p27(Kip1) stability in the G0 phase via p27(Kip1) Ser(10) phosphorylation, which is a prerequisite for induction of primary cilia. Importantly, in control embryos, p27(Kip1) protein is clearly present and strongly phosphorylated on Ser(10) in cells on the quiescent ventral surface of the node. By contrast, the corresponding cells in the node of Acvr1 mutant embryos were proliferative and showed a dramatic attenuation in both p27(Kip1) protein levels and phosphorylation on Ser(10). Our data suggest that cell quiescence controlled by BMP signaling via ACVR1 is required for transient formation of nodal cilia, and provide insight into the fundamental question of how the node represents the mechanistic `node' that regulates the development of left-right symmetry in vertebrates.

Consistent left-right asymmetry cannot be established by late organizers in Xenopus unless the late organizer is a conjoined twin.

How embryos consistently orient asymmetries of the left-right (LR) axis is an intriguing question, as no macroscopic environmental cues reliably distinguish left from right. Especially unclear are the events coordinating LR patterning with the establishment of the dorsoventral (DV) axes and midline determination in early embryos. In frog embryos, consistent physiological and molecular asymmetries manifest by the second cell cleavage; however, models based on extracellular fluid flow at the node predict correct de novo asymmetry orientation during neurulation. We addressed these issues in Xenopus embryos by manipulating the timing and location of dorsal organizer induction: the primary dorsal organizer was ablated by UV irradiation, and a new organizer was induced at various locations, either early, by mechanical rotation, or late, by injection of lithium chloride (at 32 cells) or of the transcription factor XSiamois (which functions after mid-blastula transition). These embryos were then analyzed for the position of three asymmetric organs. Whereas organizers rescued before cleavage properly oriented the LR axis 90% of the time, organizers induced in any position at any time after the 32-cell stage exhibited randomized laterality. Late organizers were unable to correctly orient the LR axis even when placed back in their endogenous location. Strikingly, conjoined twins produced by late induction of ectopic organizers did have normal asymmetry. These data reveal that although correct LR orientation must occur no later than early cleavage stages in singleton embryos, a novel instructive influence from an early organizer can impose normal asymmetry upon late organizers in the same cell field.

Evolutionary origins of blastoporal expression and organizer activity of the vertebrate gastrula organizer gene lhx1 and its ancient metazoan paralog lhx3.

Expression of the LIM homeobox gene lhx1 (lim1) is specific to the vertebrate gastrula organizer. Lhx1 functions as a transcriptional regulatory core protein to exert ;organizer' activity in Xenopus embryos. Its ancient paralog, lhx3 (lim3), is expressed around the blastopore in amphioxus and ascidian, but not vertebrate, gastrulae. These two genes are thus implicated in organizer evolution, and we addressed the evolutionary origins of their blastoporal expression and organizer activity. Gene expression analysis of organisms ranging from cnidarians to chordates suggests that blastoporal expression has its evolutionary root in or before the ancestral eumetazoan for lhx1, but possibly in the ancestral chordate for lhx3, and that in the ascidian lineage, blastoporal expression of lhx1 ceased, whereas endodermal expression of lhx3 has persisted. Analysis of organizer activity using Xenopus embryos suggests that a co-factor of LIM homeodomain proteins, Ldb, has a conserved function in eumetazoans to activate Lhx1, but that Lhx1 acquired organizer activity in the bilaterian lineage, Lhx3 acquired organizer activity in the deuterostome lineage and ascidian Lhx3 acquired a specific transactivation domain to confer organizer activity on this molecule. Knockdown analysis using cnidarian embryos suggests that Lhx1 is required for chordin expression in the blastoporal region. These data suggest that Lhx1 has been playing fundamental roles in the blastoporal region since the ancestral eumetazoan arose, that it contributed as an 'original organizer gene' to the evolution of the vertebrate gastrula organizer, and that Lhx3 could be involved in the establishment of organizer gene networks.

Apc1 is required for maintenance of local brain organizers and dorsal midbrain survival.

The tumor suppressor Apc1 is an intracellular antagonist of the Wnt/beta-catenin pathway, which is vital for induction and patterning of the early vertebrate brain. However, its role in later brain development is less clear. Here, we examined the mechanisms underlying effects of an Apc1 zygotic-effect mutation on late brain development in zebrafish. Apc1 is required for maintenance of established brain subdivisions and control of local organizers such as the isthmic organizer (IsO). Caudal expansion of Fgf8 from IsO into the cerebellum is accompanied by hyperproliferation and abnormal cerebellar morphogenesis. Loss of apc1 results in reduced proliferation and apoptosis in the dorsal midbrain. Mosaic analysis shows that Apc is required cell-autonomously for maintenance of dorsal midbrain cell fate. The tectal phenotype occurs independently of Fgf8-mediated IsO function and is predominantly caused by stabilization of beta-catenin and subsequent hyperactivation of Wnt/beta-catenin signalling, which is mainly mediated through LEF1 activity. Chemical activation of the Wnt/beta-catenin in wild-type embryos during late brain maintenance stages phenocopies the IsO and tectal phenotypes of the apc mutants. These data demonstrate that Apc1-mediated restriction of Wnt/beta-catenin signalling is required for maintenance of local organizers and tectal integrity.