Main Body Axis Research Articles

Snips and Tales of Planarian Tails

The hydrolase Notum inhibits Wnt signaling to specify regeneration of head structures in planaria.

Molecular and Functional Characterizations of Gastrula Organizer Cells Derived from Human Embryonic Stem Cells

The Spemann-Mangold organizer is the structure that provides the signals, which initiate pattern formation in the developing vertebrate embryo, affecting the main body axes. Very little is known about axial induction in the gastrulating human embryo, as research is hindered by obvious ethical restrictions. Human embryonic stem cells (hESCs) are pluripotent cells derived from the pregastrula embryo that can differentiate in culture following a program similar to normal embryonic development but without pattern formation. Here, we show that in hESC-derived embryoid bodies, we can induce differentiation of cells that harbor markers and characteristics of the gastrula-organizer. Moreover, genetic labeling of these cells enabled their purification, and the discovery of a comprehensive set of their secreted proteins, cell surface receptors, and nuclear factors characteristic of the organizer. Remarkably, transplantation of cell populations enriched for the putative human organizer into frog embryos induced a secondary axis. Our research demonstrates that the human organizer can be induced in vitro and paves the way for the study of pattern formation and the initial regulation of body axis establishment in humans.

Micro-magnetic resonance imaging study of live quail embryos during embryonic development

Eggs containing live Japanese quail embryos were imaged using micro-magnetic resonance imaging (μMRI) at 24-h intervals from Day 0 to 8, the period during which the main body axis is being laid down and organogenesis is taking place. Considerable detail of non-embryonic structures such as the latebra was revealed at early stages but the embryo could only be visualized around Day 3. Three-dimensional (3D) changes in embryo length and volume were quantified and also changes in volume in the extra- and non-embryonic components. The embryo increased in length by 43% and nearly trebled in volume between Day 4 and Day 5. Although the amount of yolk remained fairly constant over the first 5 days, the amount of albumen decreases significantly and was replaced by extra-embryonic fluid (EEF). 1H longitudinal (T1) and transverse (T2) relaxation times of different regions within the eggs were determined over the first 6 days of development. The T2 measurements mirrored the changes in image intensity observed, which can be related to the aqueous protein concentrations. In addition, a comparison of the development of Day 0 to 3 quail embryos exposed to radiofrequency (rf) pulses, 7 T static magnetic fields and magnetic field gradients for an average of 7 h with the development of control embryos did not reveal any gross changes, thus confirming that μMRI is a suitable tool for following the development of live avian embryos over time from the earliest stages.

Rapid image recognition of body parts scanned in computed tomography datasets

Automatic CT dataset classification is important to efficiently create reliable database annotations, especially when large collections of scans must be analyzed. An automated segmentation and labeling algorithm was developed based on a fast patient segmentation and extraction of statistical density class features from the CT data. The method also delivers classifications of image noise level and patient size. The approach is based on image information only and uses an approximate patient contour detection and statistical features of the density distribution. These are obtained from a slice-wise analysis of the areas filled by various materials related to certain density classes and the spatial spread of each class. The resulting families of curves are subsequently classified using rules derived from knowledge about features of the human anatomy. The method was successfully applied to more than 5,000 CT datasets. Evaluation was performed via expert visual inspection of screenshots showing classification results and detected characteristic positions along the main body axis. Accuracy per body region was very satisfactory in the trunk (lung/liver >99.5% detection rate, presence of abdomen >97% or pelvis >95.8%) improvements are required for zoomed scans. The method performed very reliably. A test on 1,860 CT datasets collected from an oncological trial showed that the method is feasible, efficient, and is promising as an automated tool for image post-processing.

3 ANALYSIS OF HOX EXPRESSION IN BOVINE OOCYTES AND EARLY EMBRYOS

HOX genes encode transcription factors known to play a major role in patterning the main body axis of vertebrate embryos from the gastrulation stage onward. A few studies have provided evidence that some HOX genes might be expressed before implantation in mammalian embryos. Translation of maternally inherited transcripts is regulated by modifications of the poly(A) tail length until embryonic genome activation (EGA), occurring during the 4th cell cycle in the bovine. The objective of this work was to establish the expression pattern of various HOX genes and to study the polyadenylation of their transcripts during oocyte maturation and early embryonic development. Pools of 20 bovine oocytes before and after in vitro maturation and 20 in vitro-produced embryos at different stages of development up to the blastocyst stage were collected. Three to 12 pools were used for each stage. RNA was extracted and reverse transcribed (RT) using random hexamers. Quantitative real-time PCR (qPCR) was performed to establish expression profiles of 4 HOX genes: HOXD1, HOXA3, HOXB9, and HOXC9. Two distinct patterns of expression were observed. First, relative amounts of HOXD1, HOXA3, and HOXC9 were lower in morulae and blastocysts than in oocytes. On the other hand, relative expression of HOXB9 increased between the 5 to 8 cell stage and the morula stage (Mann-Whitney, P < 0.05). Those expression patterns were not modified when embryos were cultured in presence of α-amanitin, a RNA polymerase II inhibitor, indicating the maternal origin of the transcripts until EGA. Total amount of mRNAs, estimated by RT-qPCR with random hexamers, was stable for all studied genes during oocyte maturation. The relative amount of polyadenylated GAPDH mRNAs, estimated by RT-qPCR with poly(dT), decreased greatly in mature oocytes compared with immature oocytes indicating massive deadenylation of those transcripts. The relative amount of polyadenylated HOXC9 transcripts decreased slightly but significantly during oocyte maturation (Mann-Whitney, P < 0.05).The relative amount of polyadenylatedm RNAs corresponding to HOXD1, HOXA3, and HOXB9 was stable during oocyte maturation. This indicates that those transcripts escape the default deadenylation pathways followed by housekeeping genes. This experiment has been repeated 3 to 4 times. In conclusion, we confirmed the presence of HOXD1, HOXA3, HOXB9, and HOXC9 transcripts in bovine oocytes and early-stage embryos. Their role during oocyte maturation and the first stages of embryonic development will be investigated through loss of function studies. This work is funded by the Fonds National de la Recherche Scientifique (Belgium).

Models for the generation and interpretation of gradients.

Source regions for morphogen gradients-organizing regions-can be generated if a local self-enhancing reaction is coupled with a long-ranging reaction that acts antagonistically. Resulting gradients can be translated into patterns of stable gene activities using genes whose products have a positive feedback on the activation on themselves. If several autoregulatory genes compete with each other for activity, cells make an unequivocal choice. Although the signal consists of a smoothly graded distribution, the all-or-nothing response of the cells leads to regions of differently determined cells that are delimited by sharp borders. In some systems, it is not the absolute but the relative level of a gradient that matters. The sequence of head, tentacles, and foot formation in hydra is controlled by a head activation gradient and is an example of this widely used but conceptually rather neglected mode. For subpatterns such as legs and wings, different "compartments" cooperate to produce new signaling substances. Here, morphogen production is restricted to the common borders or where they intersect. The model accounts for the formation of substructures in pairs at the correct positions within the embryo and for the correct orientation and handedness with respect to the main body axes.

The Bilaterally Asymmetrical Larval Form ofStomopneustes variolaris(Lamarck)

This study describes the echinopluteus and juveniles of the Indo-Pacific echinoid Stomopneustes variolaris. Late 4-armed larvae had left postoral arms that were longer and more deeply red pigmented than the right arms. Two weeks into development, the sixth pair of arms, the posterolaterals, began to form as these larvae achieved an arbacioid form. The right posterolateral arm grew long, was heavily pigmented at the tip, and was oriented perpendicularly or obliquely to the main body axis. The left posterolateral arm was relatively short, with little pigment. Two of several hundred larvae examined showed different patterns. One, with a juvenile rudiment on the right side, had arms that were a mirror image of those of typical larvae. A second larva, without a rudiment, had equal postoral arms and long, deeply pigmented posterolateral arms. These patterns suggest a developmental link between the asymmetry of the larval arms and the formation of the juvenile rudiment. Adult Stomopneustes also often showed a fixed asymmetry, with the test higher and spines shorter on the side toward interambulacrum 3 and the test lower and spines longer on the opposite side (ambulacrum I). Cleared 1- and 2-day juveniles did not show any obvious asymmetry in the location of apical plates that form from the larval spicules, so there is no evidence for a morphological link between asymmetrical larvae and adults.

Uncoupling Time and Space in the Collinear Regulation of Hox Genes

During development of the vertebrate body axis, Hox genes are transcribed sequentially, in both time and space, following their relative positions within their genomic clusters. Analyses of animal genomes support the idea that Hox gene clustering is essential for coordinating the various times of gene activations. However, the eventual collinear ordering of the gene specific transcript domains in space does not always require genomic clustering. We analyzed these complex regulatory relationships by using mutant alleles at the mouse HoxD locus, including one that splits the cluster into two pieces. We show that both positive and negative regulatory influences, located on either side of the cluster, control an early phase of collinear expression in the trunk. Interestingly, this early phase does not systematically impact upon the subsequent expression patterns along the main body axis, indicating that the mechanism underlying temporal collinearity is distinct from those acting during the second phase. We discuss the potential functions and evolutionary origins of these mechanisms, as well as their relationship with similar processes at work during limb development.

TwoHoxc6transcripts are differentially expressed and regulate primary neurogenesis inXenopus laevis

Hox genes are key players in defining positional information along the main body axis of vertebrate embryos. In Xenopus laevis, Hoxc6 was the first homeobox gene isolated. It encodes two isoforms. We analyzed in detail their spatial and temporal expression pattern during early development. One major expression domain of both isoforms is the spinal cord portion of the neural tube. Within the spinal cord and its populations of primary neurons, Hox genes have been found to play a crucial role for defining positional information. Here we report that a loss-of-function of either one of the Hoxc6 products does not affect neural induction, the expression of general neural markers is not modified. However, Hoxc6 does widely affect the formation of primary neurons within the developing neural tissue. Manipulations of Hoxc6 expression severly changes the expression of the neuronal markers N-tubulin and Islet-1. Formation of primary neurons and formation of cranial nerves are affected. Hence, Hoxc6 functions are not restricted to the expected role in anterior-posterior pattern formation, but they also regulate N-tubulin, thereby having an effect on the initial formation of primary neurons in Xenopus laevis embryos.

Generating asymmetries in the early vertebrate embryo: the role of the Cerberus-like family

One fundamental aspect of vertebrate embryonic development is the formation of the body plan. For this process, asymmetries have to be generated during early stages of development along the three main body axes: Anterior-Posterior, Dorso-Ventral and Left-Right. We have been studying the role of a novel class of molecules, the Cerberus/Dan gene family. These are dedicated secreted antagonists of three major signaling pathways: Nodal, BMP and Wnt. Our studies contribute to the current view that the fine tuning of signaling is controlled by a set of inhibitory molecules rather than by activators. In this context, the Cerberus-like molecules emerge as key players in the regulation and generation of asymmetries in the early vertebrate embryo.

Hunting for “factor X”: the genetic basis of segmental mismatch

Defending an evo-devo approach to the study of evolution in the pages of this journal may appear to be an unnecessary exercise. However, there are some case studies which, better than others, illustrate the necessity and the potential of considering developmental causes for explaining the evolution of organisms’ body architecture. An example involving the evolution of segmentation in arthropods is the recent study by Janssen et al. (2008). A zoology textbook would typically define a segmented animal as one whose body is composed of a series of modules (segments) that, more or less morphologically differentiated, are aligned along the main body axis. However, there are segmented animals in which such a modular composition is hardly recognizable, because different serial structures along the same axis of the same organism are repeated in discordant arrangement. This condition, termed ‘‘segmental mismatch,’’ characterizes the trunk of many arthropod taxa, in particular myriapods (millipedes, centipedes, and their allies), where the

Ctenophores

Ctenophores

Early evolution of symmetry and polarity in metazoan body plans

The early diverging metazoan lineages have highly disparate adult body plan geometries, which can be characterised in terms of five major types of symmetry (asymmetrical, spherical, cylindrical, n-radial, bilateral). Patterns of evolutionary changes in symmetry types and the homology of body axes across lineages are discussed here by confronting evidence from comparative anatomy, phylogeny, genomics and evo-devo. The conventional scenario, postulating a graded complexification from asymmetry to radial and finally bilateral symmetry, is considered untenable. Cylindrical symmetry is likely to be the ancestral type from which derived all remaining types through multiple convergences. Recent proposals prompted by molecular data that the bilateral anatomies of many cnidarians and of the Bilateria are homologous are clearly not supported. The Hox-based patterning system operating along the antero-posterior axis of the Bilateria does not seem to predate their divergence with the Cnidaria, but intercellular signalling systems, notably the Wnt pathway, could have been involved in generating the main body axis in the last common ancestor of the Metazoa. To cite this article: M. Manuel, C. R. Biologies 332 (2009).

Differential Expression of WOX Genes Mediates Apical-Basal Axis Formation in the Arabidopsis Embryo

Axis formation is one of the earliest patterning events in plant and animal embryogenesis. In Arabidopsis, the main axis of the embryo is evident at the asymmetric division of the zygote into a small, embryonic apical cell and a large extraembryonic basal cell. Here we show that the homeobox genes WOX2 and WOX8, which are initially coexpressed in the zygote, act as complementary cell fate regulators in the apical and basal lineage, respectively. Furthermore, WOX8 expression in the basal cell lineage is required for WOX2 expression and normal development of the proembryo, suggesting an inductive mechanism. The identified WOX cascade is required for normal expression of a reporter gene of the auxin efflux carrier PIN1 and for the formation of auxin response maxima in the proembryo. Thus, our results link the spatial separation of WOX transcription factors to localized auxin response and the formation of the main body axis in the embryo.

ParaHox gene expression in larval and postlarval development of the polychaete Nereis virens(Annelida, Lophotrochozoa)

BackgroundTranscription factors that encode ANTP-class homeobox genes play crucial roles in determining the body plan organization and specification of different organs and tissues in bilaterian animals. The three-gene ParaHox family descends from an ancestral gene cluster that existed before the evolution of the Bilateria. All three ParaHox genes are reported from deuterostomes and lophotrochozoans, but not to date from any ecdysozoan taxa, and there is evidence that the ParaHox genes, like the related Hox genes, were ancestrally a single chromosomal cluster. However, unlike the Hox genes, there is as yet no strong evidence that the ParaHox genes are expressed in spatial and temporal order during embryogenesis.ResultsWe isolated fragments of the three Nereis virens ParaHox genes, then used these as probes for whole-mount in situ hybridization in larval and postlarval worms. In Nereis virens the ParaHox genes participate in antero-posterior patterning of ectodermal and endodermal regions of the digestive tract and are expressed in some cells in the segment ganglia. The expression of these genes occurs in larval development in accordance with the position of these cells along the main body axis and in postlarval development in accordance with the position of cells in ganglia along the antero-posterior axis of each segment. In none of these tissues does expression of the three ParaHox genes follow the rule of temporal collinearity.ConclusionIn Nereis virens the ParaHox genes are expressed during antero-posterior patterning of the digestive system (ectodermal foregut and hindgut, and endodermal midgut) of Nereis virens. These genes are also expressed during axial specification of ventral neuroectodermal cell domains, where the expression domains of each gene are re-iterated in each neuromere except for the first parapodial segment. These expression domains are probably predetermined and may be directed on the antero-posterior axis by the Hox genes, whose expression starts much earlier during embryogenesis. Our results support the hypothesis that the ParaHox genes are involved in antero-posterior patterning of the developing embryo, but they do not support the notion that these genes function only in the patterning of endodermal tissues.

From development to biodiversity—Tribolium castaneum, an insect model organism for short germband development

Insect embryogenesis is best understood in the fruit fly Drosophila. However, Drosophila embryogenesis shows evolutionary-derived features: anterior patterning is controlled by a highly derived Hox gene bicoid, the body segments form almost simultaneously and appendages develop from imaginal discs. In contrast, embryogenesis of the red flour beetle Tribolium castaneum displays typical features in anterior patterning, axis and limb formation shared with most insects, other arthropods as well as with vertebrates. Anterior patterning depends on the conserved homeobox gene orthodenticle, the main body axis elongates sequentially and limbs grow continuously starting from an appendage bud. Thus, by analysing developmental processes in the beetle at the molecular and cellular level, inferences can be made for similar processes in other arthropods. With the completion of sequencing the Tribolium genome, the door is now open for post-genomic studies such as RNA expression profiling, proteomics and functional genomics to identify beetle-specific gene circuits.

Perturbation of organogenesis by the herbicide atrazine in the amphibian Xenopus laevis.

BackgroundExposure to anthropogenic chemicals during development can disrupt the morphogenesis of organ systems. Use of the herbicide atrazine has been debated in recent years because of its implicated, but poorly characterized, effects on vertebrates. Previous studies primarily examined the effects of atrazine exposure during metamorphosis or early developmental stages of amphibians.ObjectivesWe sought to identify and characterize the susceptibility during the often-overlooked developmental stage of organ morphogenesis.MethodsWe used a static renewal experimental treatment to investigate the effects of 10, 25, and 35 mg/L atrazine from early organ morphogenesis through the onset of tadpole feeding in the aquatic amphibian model system, Xenopus laevis. We quantified malformations of the body axis, heart, and intestine, as well as apoptosis in the midbrain and pronephric kidney.ResultsWe found a significant dose-dependent increase in the percentage of atrazine-exposed tadpoles with malformations of multiple tissues including the main body axis, circulatory system, kidney, and digestive system. Incidence of apoptotic cells also increased in the both midbrain and kidney of atrazine-exposed tadpoles.ConclusionsOur results demonstrate that acute atrazine exposure (10–35 mg/L for ≤ 48 hr) during early organ morphogenesis disrupts proper organ development in an amphibian model system. The concurrent atrazine-induced apoptosis in the pronephric kidney and midbrain begins to elucidate a mechanism by which atrazine may disrupt developmental processes in nontarget organisms.

Changing Hydrozoan Bauplans by Silencing Hox-Like Genes

Regulatory genes of the Antp class have been a major factor for the invention and radiation of animal bauplans. One of the most diverse animal phyla are the Cnidaria, which are close to the root of metazoan life and which often appear in two distinct generations and a remarkable variety of body forms. Hox-like genes have been known to be involved in axial patterning in the Cnidaria and have been suspected to play roles in the genetic control of many of the observed bauplan changes. Unfortunately RNAi mediated gene silencing studies have not been satisfactory for marine invertebrate organisms thus far. No direct evidence supporting Hox-like gene induced bauplan changes in cnidarians have been documented as of yet. Herein, we report a protocol for RNAi transfection of marine invertebrates and demonstrate that knock downs of Hox-like genes in Cnidaria create substantial bauplan alterations, including the formation of multiple oral poles (“heads”) by Cnox-2 and Cnox-3 inhibition, deformation of the main body axis by Cnox-5 inhibition and duplication of tentacles by Cnox-1 inhibition. All phenotypes observed in the course of the RNAi studies were identical to those obtained by morpholino antisense oligo experiments and are reminiscent of macroevolutionary bauplan changes. The reported protocol will allow routine RNAi studies in marine invertebrates to be established.

The Wnt code: cnidarians signal the way

Cnidarians are the simplest metazoans with a nervous system. They are well known for their regeneration capacity, which is based on the restoration of a signalling centre (organizer). Recent work has identified the canonical Wnt pathway in the freshwater polyp Hydra, where it acts in organizer formation and regeneration. Wnt signalling is also essential for cnidarian embryogenesis. In the sea anemone Nematostella vectensis 11 of the 12 known wnt gene subfamilies were identified. Different wnt genes exhibit serial and overlapping expression domains along the oral-aboral axis of the embryo (the 'wnt code'). This is reminiscent of the hox code (cluster) in bilaterian embryogenesis that is, however, absent in cnidarians. It is proposed that the common ancestor of cnidarians and bilaterians invented a set of wnt genes that patterned the ancient main body axis. Major antagonists of Wnt ligands (e.g. Dkk 1/2/4) that were previously known only from chordates, are also present in cnidarians and exhibit a similar conserved function. The unexpectedly high level of genetic complexity of wnt genes evolved in early multi-cellular animals about 650 Myr ago and suggests a radical expansion of the genetic repertoire, concurrent with the evolution of multi-cellularity and the diversification of eumetazoan body plans.

Genomic Evolution of Hox Gene Clusters

The family of Hox genes, which number 4 to 48 per genome depending on the animal, control morphologies on the main body axis of nearly all metazoans. The conventional wisdom is that Hox genes are arranged in chromosomal clusters in colinear order with their expression patterns on the body axis. However, recent evidence has shown that Hox gene clusters are fragmented, reduced, or expanded in many animals-findings that correlate with interesting morphological changes in evolution. Hox gene clusters also contain many noncoding RNAs, such as intergenic regulatory transcripts and evolutionarily conserved microRNAs, some of whose developmental functions have recently been explored.