Major Body Axes Research Articles

Wnt signaling in whole-body regeneration.

Regeneration abilities are widespread among animals and select species can restore any body parts removed by wounds that sever the major body axes. This capability of whole-body regeneration as exemplified in flatworm planarians, Acoels, and Cnidarians involves initial responses to injury, the assessment of wound site polarization, determination of missing tissue and programming of blastema fate, and patterned outgrowth to restore axis content and proportionality. Wnt signaling drives many shared and conserved aspects of the biology of whole-body regeneration in the planarian species Schmidtea mediterranea and Dugesia japonica, in the Acoel Hofstenia miamia, and in Cnidarians Hydra and Nematostella. These overlapping mechanisms suggest whole-body regeneration might be an ancestral property across diverse animal taxa.

Regulation of axial elongation by Cdx

The primordia of the post-otic mouse embryo forms largely from a bipotential cell population containing neuromesodermal progenitors (NMP) which reside in the tail bud and contribute to the elaboration of the major body axis after gastrulation. The mechanisms by which the NMP population is both maintained and subsequently directed down mesodermal and neural lineages is incompletely understood. The vertebrate transcription factor Cdx2, is essential for axial elongation and has been implicated in maintaining the NMP niche and in specification of NMP derivatives. To better understand the role of the Cdx family in axial elongation, we employed a conditional mutant allele which evokes total loss of Cdx function, and enriched for tail bud progenitors through the use of a Pax2-GFP transgenic reporter. Using this approach, we identified 349 Cdx-dependent genes by RNA sequencing (RNA-seq). From these, Gene Ontology and chromatin immunoprecipitation analysis further revealed a number of putative direct Cdx candidate target genes implicated in axial elongation, including Sp8, Isl1, Evx1, Zic3 and Nr2f1. Additional analysis of available single-cell RNA-seq data from mouse tail buds revealed the co-expression of Sp8, Isl1, Evx1 and Zic3 with Cdx2 in putative NMP cells, while Nr2f1 was excluded from this population. These findings identify a number of novel Cdx targets and provide further insight into the critical roles for Cdx in elaborating the post-otic embryo.

Sequential in cis mutagenesis in vivo reveals various functions for CTCF sites at the mouse HoxD cluster.

Mammalian Hox gene clusters contain a range of CTCF binding sites. In addition to their importance in organizing a TAD border, which isolates the most posterior genes from the rest of the cluster, the positions and orientations of these sites suggest that CTCF may be instrumental in the selection of various subsets of contiguous genes, which are targets of distinct remote enhancers located in the flanking regulatory landscapes. We examined this possibility by producing an allelic series of cumulative in cis mutations in these sites, up to the abrogation of CTCF binding in the five sites located on one side of the TAD border. In the most impactful alleles, the global chromatin architecture of the locus was modified, yet not drastically, illustrating that CTCF sites located on one side of a strong TAD border are sufficient to organize at least part of this insulation. Spatial colinearity in the expression of these genes along the major body axis was nevertheless maintained, despite abnormal expression boundaries. In contrast, strong effects were scored in the selection of target genes responding to particular enhancers, leading to the misregulation of Hoxd genes in specific structures. Altogether, while most enhancer-promoter interactions can occur in the absence of this series of CTCF sites, the binding of CTCF in the Hox cluster is required to properly transform a rather unprecise process into a highly discriminative mechanism of interactions, which is translated into various patterns of transcription accompanied by the distinctive chromatin topology found at this locus. Our allelic series also allowed us to reveal the distinct functional contributions for CTCF sites within this Hox cluster, some acting as insulator elements, others being necessary to anchor or stabilize enhancer-promoter interactions, and some doing both, whereas they all together contribute to the formation of a TAD border. This variety of tasks may explain the amazing evolutionary conservation in the distribution of these sites among paralogous Hox clusters or between various vertebrates.

Embryonic development in the acoel Hofstenia miamia.

Acoels are marine worms that belong to the phylum Xenacoelomorpha, a deep-diverging bilaterian lineage. This makes acoels an attractive system for studying the evolution of major bilaterian traits. Thus far, acoel development has not been described in detail at the morphological and transcriptomic levels in a species in which functional genetic studies are possible. We present a set of developmental landmarks for embryogenesis in the highly regenerative acoel Hofstenia miamia. We generated a developmental staging atlas from zygote to hatched worm based on gross morphology, with accompanying bulk transcriptome data. Hofstenia embryos undergo a stereotyped cleavage program known as duet cleavage, which results in two large vegetal pole 'macromeres' and numerous small animal pole 'micromeres'. These macromeres become internalized as micromere progeny proliferate and move vegetally. We also noted a second, previously undescribed, cell-internalization event at the animal pole, following which we detected major body axes and tissues corresponding to all three germ layers. Our work on Hofstenia embryos provides a resource for mechanistic investigations of acoel development, which will yield insights into the evolution of bilaterian development and regeneration.

A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish.

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.

Gastruloids generated without exogenous Wnt activation develop anterior neural tissues

SummaryWhen stimulated with a pulse from an exogenous WNT pathway activator, small aggregates of mouse embryonic stem cells (ESCs) can undergo embryo-like axial morphogenesis and patterning along the three major body axes. However, these structures, called gastruloids, currently lack the anterior embryonic regions, such as those belonging to the brain. Here, we describe an approach to generate gastruloids that have a more complete antero-posterior development. We used hydrogel microwell arrays to promote the robust derivation of mouse ESCs into post-implantation epiblast-like (EPI) aggregates in a reproducible and scalable manner. These EPI aggregates break symmetry and axially elongate without external chemical stimulation. Inhibition of WNT signaling in early stages of development leads to the formation of gastruloids with anterior neural tissues. Thus, we provide a new tool to study the development of the mouse after implantation in vitro, especially the formation of anterior neural regions.

Neural architecture and regeneration in the acoel Hofstenia miamia.

The origin of bilateral symmetry, a major transition in animal evolution, coincided with the evolution of organized nervous systems that show regionalization along major body axes. Studies of Xenacoelomorpha, the likely outgroup lineage to all other animals with bilateral symmetry, can inform the evolutionary history of animal nervous systems. Here, we characterized the neural anatomy of the acoel Hofstenia miamia. Our analysis of transcriptomic data uncovered orthologues of enzymes for all major neurotransmitter synthesis pathways. Expression patterns of these enzymes revealed the presence of a nerve net and an anterior condensation of neural cells. The anterior condensation was layered, containing several cell types with distinct molecular identities organized in spatially distinct territories. Using these anterior cell types and structures as landmarks, we obtained a detailed timeline for regeneration of the H. miamia nervous system, showing that the anterior condensation is restored by eight days after amputation. Our work detailing neural anatomy in H. miamia will enable mechanistic studies of neural cell type diversity and regeneration and provide insight into the evolution of these processes.

Around the clock: gradient shape and noise impact the evolution of oscillatory segmentation dynamics

BackgroundSegmentation, the subdivision of the major body axis into repeated elements, is considered one of the major evolutionary innovations in bilaterian animals. In all three segmented animal clades, the predominant segmentation mechanism is sequential segmentation, where segments are generated one by one in anterior–posterior order from a posterior undifferentiated zone. In vertebrates and arthropods, sequential segmentation is thought to arise from a clock-and-wavefront-type mechanism, where oscillations in the posterior growth zone are transformed into a segmental prepattern in the anterior by a receding wavefront. Previous evo-devo simulation studies have demonstrated that this segmentation type repeatedly arises, supporting the idea of parallel evolutionary origins in these animal clades. Sequential segmentation has been studied most extensively in vertebrates, where travelling waves have been observed that reflect the slowing down of oscillations prior to their cessation and where these oscillations involve a highly complex regulatory network. It is currently unclear under which conditions this oscillator complexity and slowing should be expected to evolve, how they are related and to what extent similar properties should be expected for sequential segmentation in other animal species.ResultsTo investigate these questions, we extend a previously developed computational model for the evolution of segmentation. We vary the slope of the posterior morphogen gradient and the strength of gene expression noise. We find that compared to a shallow gradient, a steep morphogen gradient allows for faster evolution and evolved oscillator networks are simpler. Furthermore, under steep gradients, damped oscillators often evolve, whereas shallow gradients appear to require persistent oscillators which are regularly accompanied by travelling waves, indicative of a frequency gradient. We show that gene expression noise increases the likelihood of evolving persistent oscillators under steep gradients and of evolving frequency gradients under shallow gradients. Surprisingly, we find that the evolutions of oscillator complexity and travelling waves are not correlated, suggesting that these properties may have evolved separately.ConclusionsBased on our findings, we suggest that travelling waves may have evolved in response to shallow morphogen gradients and gene expression noise. These two factors may thus also be responsible for the observed differences between different species within both the arthropod and chordate phyla.

Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids.

The emergence of multiple axes is an essential element in the establishment of the mammalian body plan. This process takes place shortly after implantation of the embryo within the uterus and relies on the activity of gene regulatory networks that coordinate transcription in space and time. Whereas genetic approaches have revealed important aspects of these processes1, a mechanistic understanding is hampered by the poor experimental accessibility of early post-implantation stages. Here we show that small aggregates of mouse embryonic stem cells (ESCs), whenstimulated to undergo gastrulation-like events and elongation in vitro, can organize a post-occipital pattern of neural, mesodermal and endodermal derivatives that mimic embryonic spatial and temporal gene expression. The establishment of the three major body axes in these 'gastruloids'2,3 suggests that the mechanisms involved are interdependent. Specifically, gastruloids display the hallmarks of axial gene regulatory systems as exemplified by the implementation of collinear Hox transcriptional patterns along an extending antero-posterior axis. These results reveal an unanticipated self-organizing capacity of aggregated ESCs and suggest that gastruloids could be used as a complementary system to study early developmental events in the mammalian embryo.

Evolution of the bilaterian mouth and anus

It is widely held that the bilaterian tubular gut with mouth and anus evolved from a simple gut with one major gastric opening. However, there is no consensus on how this happened. Did the single gastric opening evolve into a mouth, with the anus forming elsewhere in the body (protostomy), or did it evolve into an anus, with the mouth forming elsewhere (deuterostomy), or did it evolve into both mouth and anus (amphistomy)? These questions are addressed by the comparison of developmental fates of the blastopore, the opening of the embryonic gut, in diverse animals that live today. Here we review comparative data on the identity and fate of blastoporal tissue, investigate how the formation of the through-gut relates to the major body axes, and discuss to what extent evolutionary scenarios are consistent with these data. Available evidence indicates that stem bilaterians had a slit-like gastric opening that was partially closed in subsequent evolution, leaving open the anus and most likely also the mouth, which would favour amphistomy. We discuss remaining difficulties, and outline directions for future research.

Coral development: from classical embryology to molecular control.

The phylum Cnidaria is the closest outgroup to the triploblastic metazoans and as such offers unique insights into evolutionary questions at several levels. In the post-genomic era, a knowledge of the gene complement of representative cnidarians will be important for understanding the relationship between the expansion of gene families and the evolution of morphological complexity among more highly evolved metazoans. Studies of cnidarian development and its molecular control will provide information about the origins of the major bilaterian body axes, the origin of the third tissue layer, the mesoderm, and the evolution of nervous system patterning. We are studying the cnidarian Acropora millepora, a reef building scleractinian coral, and a member of the basal cnidarian class, the Anthozoa. We review ourwork on descriptive embryology and studies of selected transcription factor gene families, where our knowledge from Acropora is particularly advanced relative to other cnidarians. We also describe a recent preliminary whole genome initiative, a coral EST database.

Evidence-Based Theory for Integrated Genome Regulation of Ontogeny--An Unprecedented Role of Nuclear FGFR1 Signaling.

Genetic experiments have positioned the fgfr1 gene at the top of the gene hierarchy that governs gastrulation, as well as the subsequent development of the major body axes, nervous system, muscles, and bones, by affecting downstream genes that control the cell cycle, pluripotency, and differentiation, as well as microRNAs. Studies show that this regulation is executed by a single protein, the nuclear isoform of FGFR1 (nFGFR1), which integrates signals from development‐initiating factors, such as retinoic acid (RA), and operates at the interface of genomic and epigenomic information. nFGFR1 cooperates with a multitude of transcriptional factors (TFs), and targets thousands of genes encoding for mRNAs, as well as miRNAs in top ontogenic networks. nFGFR1 binds to the promoters of ancient proto‐oncogenes and tumor suppressor genes, in addition to binding to metazoan morphogens that delineate body axes, and construct the nervous system, as well as mesodermal and endodermal tissues. The discovery of pan‐ontogenic gene programming by integrative nuclear FGFR1 signaling (INFS) impacts our understanding of ontogeny, as well as developmental pathologies, and holds new promise for reconstructive medicine, and cancer therapy. J. Cell. Physiol. 231: 1199–1218, 2016. © 2016 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.

Only accessible information is useful: insights from gradient-mediated patterning.

Information theory is gaining popularity as a tool to characterize performance of biological systems. However, information is commonly quantified without reference to whether or how a system could extract and use it; as a result, information-theoretic quantities are easily misinterpreted. Here, we take the example of pattern-forming developmental systems which are commonly structured as cascades of sequential gene expression steps. Such a multi-tiered structure appears to constitute sub-optimal use of the positional information provided by the input morphogen because noise is added at each tier. However, one must distinguish between the total information in a morphogen and information that can be usefully extracted and interpreted by downstream elements. We demonstrate that quantifying the information that is accessible to the system naturally explains the prevalence of multi-tiered network architectures as a consequence of the noise inherent to the control of gene expression. We support our argument with empirical observations from patterning along the major body axis of the fruit fly embryo. We use this example to highlight the limitations of the standard information-theoretic characterization of biological signalling, which are frequently de-emphasized, and illustrate how they can be resolved.

An in vitro gastrulation model recapitulates the morphogenetic impact of pharmacological inhibitors of developmental signaling pathways.

Certain chemical agents act as teratogens, causing birth defects and fetal deaths when pregnant women are exposed to them. The establishment of in vitro models that recapitulate crucial embryonic events is therefore vital to facilitate screening of potential teratogens. Previously, we created a three-dimensional culture method for mouse P19C5 embryonal carcinoma stem cells that, when cultured as embryoid bodies, display elongation morphogenesis resembling gastrulation, which is the critical event resulting in the germ layers and major body axes. Determination of how well this in vitro morphogenesis represents in vivo gastrulation is essential to assess its applicability as well as to identify limitations of the model for detecting teratogenic agents. Here, we investigated the morphological and molecular characteristics of P19C5 morphogenesis using pharmacological agents that are known to cause abnormal patterning in the embryo in vivo by inhibiting major developmental signaling--e.g., involving Wnt, Nodal, Bone morphogenic protein (Bmp), Fibroblast growth factor (Fgf), Retinoic acid, Notch, and Hedgehog pathways. Inhibitors of Wnt, Nodal, Bmp, Fgf, and Retinoic acid signaling caused distinct changes in P19C5 morphogenesis that were quantifiable using morphometric parameters. These five inhibitors, plus the Notch inhibitor, also altered temporal expression profiles of developmental regulator genes in a manner consistent with the in vivo roles of the corresponding signaling pathways. In contrast, the Hedgehog inhibitor did not have any impact on the process, suggesting an absence of active Hedgehog signaling in these embryoid bodies. These results indicate that the P19C5 in vitro gastrulation model is a promising tool to screen for teratogenic agents that interfere with many of the key developmental signals.

Long-range gap junctional signaling controls oncogene-mediated tumorigenesis in Xenopus laevis embryos.

In addition to the immediate microenvironment, long-range signaling may be an important component of cancer. Molecular-genetic analyses have implicated gap junctions—key mediators of cell-cell communication—in carcinogenesis. We recently showed that the resting voltage potential of distant cell groups is a key determinant of metastatic transformation and tumor induction. Here, we show in the Xenopus laevis model that gap junctional communication (GJC) is a modulator of the long-range bioelectric signaling that regulates tumor formation. Genetic disruption of GJC taking place within tumors, within remote host tissues, or between the host and tumors significantly lowers the incidence of tumors induced by KRAS mutations. The most pronounced suppression of tumor incidence was observed upon GJC disruption taking place farther away from oncogene-expressing cells, revealing a role for GJC in distant cells in the control of tumor growth. In contrast, enhanced GJC communication through the overexpression of wild-type connexin Cx26 increased tumor incidence. Our data confirm a role for GJC in tumorigenesis, and reveal that this effect is non-local. Based on these results and on published data on movement of ions through GJs, we present a quantitative model linking the GJC coupling and bioelectrical state of cells to the ability of oncogenes to initiate tumorigenesis. When integrated with data on endogenous bioelectric signaling during left-right patterning, the model predicts differential tumor incidence outcomes depending on the spatial configurations of gap junction paths relative to tumor location and major anatomical body axes. Testing these predictions, we found that the strongest influence of GJ modulation on tumor suppression by hyperpolarization occurred along the embryonic left-right axis. Together, these data reveal new, long-range aspects of cancer control by the host's physiological parameters.

A novel role for Ascl1 in the regulation of mesendoderm formation via HDAC-dependent antagonism of VegT.

Maternally expressed proteins function in vertebrates to establish the major body axes of the embryo and to establish a pre-pattern that sets the stage for later-acting zygotic signals. This pre-patterning drives the propensity of Xenopus animal cap cells to adopt neural fates under various experimental conditions. Previous studies found that the maternally expressed transcription factor, encoded by the Xenopus achaete scute-like gene ascl1, is enriched at the animal pole. Asc1l is a bHLH protein involved in neural development, but its maternal function has not been studied. Here, we performed a series of gain- and loss-of-function experiments on maternal ascl1, and present three novel findings. First, Ascl1 is a repressor of mesendoderm induced by VegT, but not of Nodal-induced mesendoderm. Second, a previously uncharacterized N-terminal domain of Ascl1 interacts with HDAC1 to inhibit mesendoderm gene expression. This N-terminal domain is dispensable for its neurogenic function, indicating that Ascl1 acts by different mechanisms at different times. Ascl1-mediated repression of mesendoderm genes was dependent on HDAC activity and accompanied by histone deacetylation in the promoter regions of VegT targets. Finally, maternal Ascl1 is required for animal cap cells to retain their competence to adopt neural fates. These results establish maternal Asc1l as a key factor in establishing pre-patterning of the early embryo, acting in opposition to VegT and biasing the animal pole to adopt neural fates. The data presented here significantly extend our understanding of early embryonic pattern formation.

A Genetic Screen Based onin VivoRNA Imaging Reveals Centrosome-Independent Mechanisms for LocalizinggurkenTranscripts inDrosophila

We have screened chromosome arm 3L for ethyl methanesulfonate−induced mutations that disrupt localization of fluorescently labeled gurken (grk) messenger (m)RNA, whose transport along microtubules establishes both major body axes of the developing Drosophila oocyte. Rapid identification of causative mutations by single-nucleotide polymorphism recombinational mapping and whole-genomic sequencing allowed us to define nine complementation groups affecting grk mRNA localization and other aspects of oogenesis, including alleles of elg1, scaf6, quemao, nudE, Tsc2/gigas, rasp, and Chd5/Wrb, and several null alleles of the armitage Piwi-pathway gene. Analysis of a newly induced kinesin light chain allele shows that kinesin motor activity is required for both efficient grk mRNA localization and oocyte centrosome integrity. We also show that initiation of the dorsoanterior localization of grk mRNA precedes centrosome localization, suggesting that microtubule self-organization contributes to breaking axial symmetry to generate a unique dorsoventral axis.

Hox proteins drive cell segregation and non-autonomous apical remodelling during hindbrain segmentation.

Hox genes encode a conserved family of homeodomain transcription factors regulating development along the major body axis. During embryogenesis, Hox proteins are expressed in segment-specific patterns and control numerous different segment-specific cell fates. It has been unclear, however, whether Hox proteins drive the epithelial cell segregation mechanism that is thought to initiate the segmentation process. Here, we investigate the role of vertebrate Hox proteins during the partitioning of the developing hindbrain into lineage-restricted units called rhombomeres. Loss-of-function mutants and ectopic expression assays reveal that Hoxb4 and its paralogue Hoxd4 are necessary and sufficient for cell segregation, and for the most caudal rhombomere boundary (r6/r7). Hox4 proteins regulate Eph/ephrins and other cell-surface proteins, and can function in a non-cell-autonomous manner to induce apical cell enlargement on both sides of their expression border. Similarly, other Hox proteins expressed at more rostral rhombomere interfaces can also regulate Eph/ephrins, induce apical remodelling and drive cell segregation in ectopic expression assays. However, Krox20, a key segmentation factor expressed in odd rhombomeres (r3 and r5), can largely override Hox proteins at the level of regulation of a cell surface target, Epha4. This study suggests that most, if not all, Hox proteins share a common potential to induce cell segregation but in some contexts this is masked or modulated by other transcription factors.

Components of the dorsal-ventral pathway also contribute to anterior-posterior patterning in honeybee embryos (Apis mellifera)

BackgroundA key early step in embryogenesis is the establishment of the major body axes; the dorsal-ventral (DV) and anterior-posterior (AP) axes. Determination of these axes in some insects requires the function of different sets of signalling pathways for each axis. Patterning across the DV axis requires interaction between the Toll and Dpp/TGF-β pathways, whereas patterning across the AP axis requires gradients of bicoid/orthodenticle proteins and the actions of a hierarchy of gene transcription factors. We examined the expression and function of Toll and Dpp signalling during honeybee embryogenesis to assess to the role of these genes in DV patterning.ResultsPathway components that are required for dorsal specification in Drosophila are expressed in an AP-restricted pattern in the honeybee embryo, including Dpp and its receptor Tkv. Components of the Toll pathway are expressed in a more conserved pattern along the ventral axis of the embryo. Late-stage embryos from RNA interference (RNAi) knockdown of Toll and Dpp pathways had both DV and AP patterning defects, confirmed by staining with Am-sna, Am-zen, Am-eve, and Am-twi at earlier stages. We also identified two orthologues of dorsal in the honeybee genome, with one being expressed during embryogenesis and having a minor role in axis patterning, as determined by RNAi and the other expressed during oogenesis.ConclusionsWe found that early acting pathways (Toll and Dpp) are involved not only in DV patterning but also AP patterning in honeybee embryogenesis. Changes to the expression patterns and function of these genes may reflect evolutionary changes in the placement of the extra-embryonic membranes during embryogenesis with respect to the AP and DV axes.

Direct interaction between two actin nucleators is required in Drosophila oogenesis

Controlled actin assembly is crucial to a wide variety of cellular processes, including polarity establishment during early development. The recently discovered actin mesh, a structure that traverses the Drosophila oocyte during mid-oogenesis, is essential for proper establishment of the major body axes. Genetic experiments indicate that at least two proteins, Spire (Spir) and Cappuccino (Capu), are required to build this mesh. The spire and cappuccino genetic loci were first identified as maternal effect genes in Drosophila. Mutation in either locus results in the same phenotypes, including absence of the mesh, linking them functionally. Both proteins nucleate actin filaments. Spir and Capu also interact directly with each other in vitro, suggesting a novel synergistic mode of regulating actin. In order to understand how and why proteins with similar biochemical activity would be required in the same biological pathway, genetic experiments were designed to test whether a direct interaction between Spir and Capu is required during oogenesis. Indeed, data in this study indicate that Spir and Capu must interact directly with one another and then separate to function properly. Furthermore, these actin regulators are controlled by a combination of mechanisms, including interaction with one another, functional inhibition and regulation of their protein levels. Finally, this work demonstrates for the first time in a multicellular organism that the ability of a formin to assemble actin filaments is required for a specific structure.