Towards a synthetic view of axis specification mechanisms in vertebrates: insights from the dogfish

The genetic mechanisms, which control axis specification, apparently extensively diverge across vertebrates. In amphibians and teleosts, they are tightly linked to the establishment of an early dorso-ventral polarity. This polarity has no equivalent in amniotes, which unlike the former, retain a considerable plasticity for their site of axis formation until blastula stages and rely on signals secreted by extra-embryonic tissues for the establishment of their early rostro-caudal pattern. In order to better understand the links between these seemingly highly divergent mechanisms, we have used an evo-devo approach, aimed at reconstructing the gnathostome ancestral state and focussed on a chondrichthyan, the dogfish Scyliorhinus canicula. A detailed molecular characterization of the dogfish embryo at blastula and gastrula stages highlights striking similarities with all vertebrate model organisms including amniotes. It suggests the presence in the dogfish of territories homologous to the hypoblast and extra-embryonic ectoderm of the latter, which may therefore reflect the primitive condition of jawed vertebrates. In the ancestral state, these territories are specified at opposite sides of an early axis of bilateral symmetry, homologous to the dorso-ventral axis of amphibians and teleosts, and aligned with the later forming embryonic axis, from head to tail. Amniotes have diverged from this pattern through a posterior expansion of extra-embryonic ectoderm, resulting in an apparently radial symmetry at late blastula stages. These data delineate the broad outlines of the gnathostome ancestral pattern of axis specification and highlight an unexpected unity of mechanisms across jawed vertebrates. They illustrate the complementarity of comparative and genetic approaches for a comprehensive view of developmental mechanisms themselves. To cite this article: M. Coolen et al., C. R. Biologies 332 (2009).     

A conserved role for the nodal signaling pathway in the establishment of dorso-ventral and left-right axes in deuterostomes

Nodal factors play crucial roles during embryogenesis of chordates. They have been implicated in a number of developmental processes, including mesoderm and endoderm formation and patterning of the embryo along the anterior-posterior and left-right axes. We have analyzed the function of the Nodal signaling pathway during the embryogenesis of the sea urchin, a non-chordate organism. We found that Nodal signaling plays a central role in axis specification in the sea urchin, but surprisingly, its first main role appears to be in ectoderm patterning and not in specification of the endoderm and mesoderm germ layers as in vertebrates. Starting at the early blastula stage, sea urchin nodal is expressed in the presumptive oral ectoderm where it controls the formation of the oral-aboral axis. A second conserved role for nodal signaling during vertebrate evolution is its involvement in the establishment of left-right asymmetries. Sea urchin larvae exhibit profound left-right asymmetry with the formation of the adult rudiment occurring only on the left side. We found that a nodal/lefty/pitx2 gene cassette regulates left-right asymmetry in the sea urchin but that intriguingly, the expression of these genes is reversed compared to vertebrates. We have shown that Nodal signals emitted from the right ectoderm of the larva regulate the asymmetrical morphogenesis of the coelomic pouches by inhibiting rudiment formation on the right side of the larva. This result shows that the mechanisms responsible for patterning the left-right axis are conserved in echinoderms and that this role for nodal is conserved among the deuterostomes. We will discuss the implications regarding the reference axes of the sea urchin and the ancestral function of the nodal gene in the last section of this review.     

Somite patterning and segregation of different somite lineages]

The somite is a transient embryonic mesodermal structure, found only in vertebrates. In amniotes, somites give rise to the dermis of the back, to the striated skeletal muscles of the trunk and limbs and to the vertebral column and ribs. Segregation of these different lineages is linked to the establishment of two somitic polarity axes, a dorso-ventral one and a medio-lateral one. While the establishment of the former relies essentially on extrinsic cues, that of the latter obeys to both intrinsic and extrinsic mechanisms. Concerning the environmental cues regulating the establishment of both axes, somitic regionalisation results from antagonistic or combinatorial influences mediated by diffusible factors, such as Sonic Hedgehog, Wnt and Bmp-4, that act in gradients.     

Water-soluble proteins in early amphibian embryos. III. Immunoelectrophoretic analysis of the antigenic changes in the ectoderm of the early gastrula and neural plate during development

20 water-soluble antigen have been identified with the help of rabbit antisera to extracts of the early gastrula ectoderm and neural plate in Rana temporaria. All of them were also found in the early blastula embryos and unfertilized eggs. The identified antigens are characterized by a definite embryospecificity. As the development proceeds, the concentration of these antigens in the embryonic tissues decreases until the complete disappearance of corresponding immunoelectrophoretic reactions. By this characteristic all antigens under study are subdivided into four groups: I--five antigens identified at the early developmental stages only (until hatching, stage 29); II--nine antigens present up to stages 33--35; III--three antigens followed up to stages 39--40 (well formed tadpole); IV--three antigens were found at all developmental stages under study up to stages 45--47. 11 out of 20 identified antigens have antigenic similarity with the proteins of blood serum of adult amphibians. Besides, the early gastrula ectoderm contains antigens similar with those of the brain of adult amphibians.     

Regional specification during embryogenesis in the inarticulate brachiopod Discinisca.

The process of embryogenesis is described for the inarticulate brachiopod Discinisca strigata of the family Discinidae. A fate map has been constructed for the early embryo. The animal half of the egg forms the dorsal ectoderm of the apical and mantle lobes. The vegetal half forms mesoderm and endoderm and is the site of gastrulation; it also forms the ectoderm of the ventral regions of the apical and mantle lobes of the larva. The plane of the first cleavage goes through the animal-vegetal axis of the egg along the future plane of bilateral symmetry of the larva. The timing of regional specification in these embryos was examined by isolating animal, vegetal, or lateral regions at different times from the 2-cell stage through gastrulation. Animal halves isolated at the 8-cell and blastula stages formed an epithelial vesicle and did not gastrulate. When these halves were isolated from blastulae they formed the cell types typical of apical and mantle lobes. Vegetal halves isolated at all stages gastrulated and formed a more or less normal larva; the only defect these larvae had was the lack of an apical tuft, which normally forms from cells at the animal pole of the embryo. When lateral isolates were created at all developmental stages, these halves gastrulated. Cuts which separated presumptive anterior and posterior regions generated isolates at the 4-cell and blastula stages that formed essentially normal larvae; however, at the midgastrula stage these halves formed primarily anterior or posterior structures indicating that regional specification had taken place along the anterior-posterior axis. The plane of the first cleavage, which predicts the plane of bilateral symmetry, can be shifted by either changing the cleavage pattern that generates the bilateral 16-cell blastomere configuration or by isolating embryo halves prior to, or during, the 16-cell stage. These results indicate that while the plane of the first cleavage predicts the axis of bilateral symmetry, the axis is not established until the fourth cleavage. The development of Discinisca is compared to development in the inarticulate brachiopod Glottidia of the family Lingulidae and to Phoronis in the phylum Phoronida.     

From fertilization to gastrulation: axis formation in the mouse embryo.

Although much remains unknown about how the embryonic axis is laid down in the mouse, it is now clear that reciprocal interactions between the extraembryonic and embryonic lineages establish and reinforce patterning of the embryo. At early post-implantation stages, the extraembryonic ectoderm appears to impart proximal-posterior identity to the adjacent proximal epiblast, whereas the distal visceral endoderm signals to the underlying epiblast to restrict posterior identity as it moves anteriorward. At gastrulation, the visceral endoderm is necessary for specifying anterior primitive streak derivatives, which, in turn, pattern the anterior epiblast. Polarity of these extraembryonic tissues can be traced back to the blastocyst stage, where asymmetry has been linked to the point of sperm entry at fertilization.     

Planar Polarity in the Ciliated Epidermis of Xenopus Embryos

The coordinated orientation of ciliary beat in the larval epidermis of amphibians, evident in an organized streamline pattern, suggests a planar polarity of the epithelium, i.e., a polarity within the plane of the cell sheet. It has been proposed that the direction of ciliary beat is determined at mid gastrula by a gradient of a diffusible factor produced by the mesoderm. To analyze whether ectoderm in isolation can establish a uniform direction of ciliary beat, and at what stage its polarity is specified in the embryo, ectoderm of Xenopus laevis embryos of different stages was cultured in vitro on substrates. On concanavalin A, ectoderm isolated at early gastrula stages, i.e., prior to any contact with mesoderm, can autonomously coordinate the direction of ciliary beat, at least in small regions. A uniform planar polarity is expressed by ectoderm explanted from the early mid gastrula onward. On fibronectin, which promotes migration, the direction of movement correlates well with the direction of ciliary beat, and directional migration can even override the inherent polarity specified prior to explantation. Embryos which lack dorsal mesoderm nevertheless develop a highly organized streamline pattern, excluding a strict requirement for dorsal mesoderm for the determination of planar polarity. However, in spite of the early specification of planar polarity found for isolated tissue, rotated ectodermal transplants in situ can readjust their polarity in accordance with that of the host.     

β-catenin specifies the endomesoderm and defines the posterior organizer of the hemichordate Saccoglossus kowalevskii

The canonical Wnt/β-catenin pathway is a key regulator of body plan organization and axis formation in metazoans, being involved in germ layer specification, posterior growth and patterning of the anteroposterior axis. Results from animals spanning a wide phylogenetic range suggest that a unifying function of β-catenin in metazoans is to define the posterior/vegetal part of the embryo. Although the specification of vegetal territories (endoderm) by β-catenin has been demonstrated in distantly related animals (cnidarians, a protostome, echinoderms and ascidians), the definition of the posterior part of the embryo is well supported only for vertebrates and planarians. To gain insights into β-catenin functions during deuterostome evolution, we have studied the early development of the direct developing hemichordate Saccoglossus kowalevskii. We show that the zygote is polarized after fertilization along the animal-vegetal axis by cytoplasmic rearrangements resembling the ascidian vegetal contraction. This early asymmetry is translated into nuclear accumulation of β-catenin at the vegetal pole, which is necessary and sufficient to specify endomesoderm. We show that endomesoderm specification is crucial for anteroposterior axis establishment in the ectoderm. The endomesoderm secretes as yet unidentified signals that posteriorize the ectoderm, which would otherwise adopt an anterior fate. Our results point to a conserved function at the base of deuterostomes for β-catenin in germ layer specification and to a causal link in the definition of the posterior part of the embryonic ectoderm by way of activating posteriorizing endomesodermal factors. Consequently, the definition of the vegetal and the posterior regions of the embryo by β-catenin should be distinguished and carefully re-examined.     

Evolution of germ cell development in tetrapods: comparison of urodeles and amniotes

SUMMARY. The embryonic development of germ cells in tetrapods is described, focusing on groups with the inductive mode of germ cell specification. In mammals PGCs are induced early in the gastrulation process, they are internalized with future extraembryonic mesoderm in the early posterior primitive streak, and specified soon thereafter. Strong evidence indicates that a similar process occurs in turtles and some other reptiles. In amniotes, the PGCs appear well before formation of the gonad in the posterior trunk, resulting in a period in which they are located outside the embryo before their migration to the gonad. In contrast, in urodeles the PGCs appear relatively late, and throughout development maintain a position close to precursors of the somatic cells of the gonad so that migration is not required. In lampreys early development of germ cells is strikingly similar to that in urodeles, suggesting this is the primitive process. As amniotes evolved large yolky eggs and better access to nutrition, development of the posterior half of the trunk became more dependent on cell proliferation; this was followed or accompanied by a shift of early germ cell development to the equivalent of the early primitive streak. A similar process may have occurred as some basal vertebrates developed large yolky eggs.     

The ectodermal control of mesodermal patterns of differentiation in the developing chick wing

The influence of limb ectoderm on the dorso-ventral muscle and skeletal patterns in the chick wing was studied by recombining stage 14-21 limb mesoderm with the same stage ectoderm in dorso-ventrally reversed orientation. Recombinants grafted to the flank of host embryos were allowed to develop for 10 days. Fully developed wings obtained from stage 15-21 donor embryos have at their distal half d-v polarity conforming to the reversed ectoderm and proximally polarity conforming with the mesoderm. The ectodermal effect is generally observed as a bidorsal feather pattern at the autopod and an almost complete d-v reversal of muscle and skeletal patterns. In experimental wings from donor embryos younger than stage 15, the dorso-ventral pattern conforms with the polarity of the limb mesoderm. The results suggest that control of dorso-ventral polarity resides in the mesoderm until the onset of limb development at stage 15. At this stage, the ectoderm acquires dorso-ventral information which it can impose on the mesoderm.     

The zebrafish bozozok locus encodes Dharma, a homeodomain protein essential for induction of gastrula organizer and dorsoanterior embryonic structures

The dorsal gastrula organizer plays a fundamental role in establishment of the vertebrate axis. We demonstrate that the zebrafish bozozok (boz) locus is required at the blastula stages for formation of the embryonic shield, the equivalent of the gastrula organizer and expression of multiple organizer-specific genes. Furthermore, boz is essential for specification of dorsoanterior embryonic structures, including notochord, prechordal mesendoderm, floor plate and forebrain. We report that boz mutations disrupt the homeobox gene dharma. Overexpression of boz in the extraembryonic yolk syncytial layer of boz mutant embryos is sufficient for normal development of the overlying blastoderm, revealing an involvement of extraembryonic structures in anterior patterning in fish similarly to murine embryos. Epistatic analyses indicate that boz acts downstream of beta-catenin and upstream to TGF-beta signaling or in a parallel pathway. These studies provide genetic evidence for an essential function of a homeodomain protein in beta-catenin-mediated induction of the dorsal gastrula organizer and place boz at the top of a hierarchy of zygotic genes specifying the dorsal midline of a vertebrate embryo.     

The different roles of cyclinD1-CDK4 in STP and mGluR-LTD during the postnatal development in mice hippocampus area CA1

Background

The extraembryonic tissues, visceral endoderm (VE) and extraembryonic ectoderm (ExE) are known to be important for the induction of primordial germ cells (PGCs) in mice via activation of the bone morphogenetic protein (BMP) signalling pathway. We investigated whether the VE and ExE have a direct role in the specification of PGCs, or in an earlier event, namely the induction of the PGC precursors in the proximal posterior epiblast cells.

Results

We cultured embryonic day (E) 5.75 to E7.0 mouse embryos in an explant-assay with or without extraembryonic tissues. The reconstituted pieces of embryonic and extraembryonic tissues were assessed for the formation of both PGC precursors and specified PGCs. For this, Blimp1:gfp and Stella:gfp transgenic mouse lines were used to distinguish between PGC precursors and specified PGC, respectively. We observed that the VE regulates formation of an appropriate number of PGC precursors between E6.25–E7.25, but it is not essential for the subsequent specification of PGCs from the precursor cells. Furthermore, we show that the ExE has a different role from that of the VE, which is to restrict localization of PGC precursors to the posterior part of the embryo.

Conclusion

We show that the VE and ExE have distinct roles in the induction of PGC precursors, namely the formation of a normal number of PGC precursors, and their appropriate localization during early development. However, these tissues do not have a direct role during the final stages of specification of the founder population of PGCs.     

Evolution of leftward flow

The asymmetric Nodal signaling cascade as a prerequisite for asymmetric body plan specification is conserved among deuterostomes. In this review we argue that symmetry breakage by cilia-driven leftward flow presents an ancestral character of vertebrates, likely the chordate phylum and maybe all deuterostomes. In vertebrates, leftward flow occurs in a transient structure, a monociliated epithelium, which is derived from superficial mesoderm and localizes to the archenteron roof during gastrulation. The chick as an example for the highly derived birds lacks superficial mesoderm and flow. This loss should be secondary, as flow is present from fish and amphibians to mammals.     

What studies of turbellarian embryos can tell us about the evolution of development mechanisms

In spiralian embryos determination of the axes of bilateral symmetry is associated with D quadrant specification. This can occur late through equal cleavage and cell interactions (conditional specification) or by the four-cell stage through unequal cleavage and cytoplasmic localization (autonomous specification). Freeman & Lundelius (1992) suggest that in spiralian coelomates the former method is ancestral and the latter derived, with evolutionary pressure to shorten metamorphosis resulting in early D quadrant determination through unequal cleavage and appearance of adult features in the larvae. Because of the key phylogenetic position of the turbellarian platyhelminthes, understanding the method of axis specification in this group is important in evaluating the hypothesis. Polyclad development, with equal quartet spiral cleavage, is believed to represent the most primitive condition among living turbellarians and has been examined experimentally in Hoploplana inquilina. Blastomere deletions at the two and four-cell stage produce larvae that are abnormal in morphology and symmetry, indicating that early development is not regulative, and also establish that the embryo does not have an invariant cell lineage. Deletions of micromeres and macromeres at the eight-cell stage indicate that cell interactions are involved in dorso-ventral axis determination, with cross-furrow macromeres playing a more significant role than non-cross-furrow cells. The results support the idea that conditional specification is the primitive developmental mode that characterized the common ancestor of the turbellarians and spiralian coelomates. Evolutionary trends in development in polyclads and other turbellarian orders are discussed.     

Establishment of segment polarity in the ectoderm of the leech Helobdella

The segmented ectoderm and mesoderm of the leech arise via a stereotyped cell lineage from embryonic stem cells called teloblasts. Each teloblast gives rise to a column of primary blast cell daughters, and the blast cells generate descendant clones that serve as the segmental repeats of their particular teloblast lineage. We have examined the mechanism by which the leech primary blast cell clones acquire segment polarity - i.e. a fixed sequence of positional values ordered along the anteroposterior axis of the segmental repeat. In the O and P teloblast lineages, the earliest divisions of the primary blast cell segregate anterior and posterior cell fates along the anteroposterior axis. Using a laser microbeam, we ablated single cells from both o and p blast cell clones at stages when the clone was two to four cells in length. The developmental fate of the remaining cells was characterized with rhodamine-dextran lineage tracer. Twelve different progeny cells were ablated, and in every case the ablation eliminated the normal descendants of the ablated cell while having little or no detectable effect on the developmental fate of the remaining cells. This included experiments in which we specifically ablated those blast cell progeny that are known to express the engrailed gene, or their lineal precursors. These findings confirm and extend a previous study by showing that the establishment of segment polarity in the leech ectoderm is largely independent of cell interactions conveyed along the anteroposterior axis. Both intercellular signaling and engrailed expression play an important role in the segment polarity specification of the Drosophila embryo, and our findings suggest that there may be little or no conservation of this developmental mechanism between those two organisms.     

Regulative specification of ectoderm in skeleton disrupted sea urchin embryos treated with monoclonal antibody to Pl-nectin

Pl-nectin is a glycoprotein first discovered in the extracellular matrix (ECM) of Paracentrotus lividus sea urchin embryo, apically located on ectoderm and endoderm cells. The molecule has been described as functioning as an adhesive substrate for embryonic cells and its contact to ectoderm cells is essential for correct skeletogenesis. The present study was undertaken to elucidate the biochemical characteristics of Pl-nectin and to extend knowledge on its in vivo biological function. Here it is shown that the binding of mesenchyme blastula cells to Pl-nectin-coated substrates was calcium dependent, and reached its optimum at 10 mM Ca2+. Perturbation studies using monoclonal antibody (McAb) to Pl-nectin, which prevent ectoderm cell-Pl-nectin contact, show that dorsoventral axis formation and ectoderm differentiation were retarded. At later stages, embryos recovered and, even if growth and patterning of the skeleton was greatly affected, the establishment of dorsoventral asymmetry was reached. Similarly, the expression of specific ectoderm and endoderm territorial markers was achieved, although occurring with some delay. Endoderm differentiation and patterning was not obviously affected. These results suggest that both endoderm and ectoderm cells have regulative capacities and differentiation of territories is restored after a lag period. On the contrary, failure of inductive differentiation of the skeleton cannot be rescued, even though the ectoderm has recovered.     

Differential Contributions of Dorso-Ventral and Rostro-Caudal Prefrontal White Matter Tracts to Cognitive Control in Healthy Older Adults

Prefrontal cortex mediates cognitive control by means of circuitry organized along dorso-ventral and rostro-caudal axes. Along the dorso-ventral axis, ventrolateral PFC controls semantic information, whereas dorsolateral PFC encodes task rules. Along the rostro-caudal axis, anterior prefrontal cortex encodes complex rules and relationships between stimuli, whereas posterior prefrontal cortex encodes simple relationships between stimuli and behavior. Evidence of these gradients of prefrontal cortex organization has been well documented in fMRI studies, but their functional correlates have not been examined with regard to integrity of underlying white matter tracts. We hypothesized that (a) the integrity of specific white matter tracts is related to cognitive functioning in a manner consistent with the dorso-ventral and rostro-caudal organization of the prefrontal cortex, and (b) this would be particularly evident in healthy older adults. We assessed three cognitive processes that recruit the prefrontal cortex and can distinguish white matter tracts along the dorso-ventral and rostro-caudal dimensions –episodic memory, working memory, and reasoning. Correlations between cognition and fractional anisotropy as well as fiber tractography revealed: (a) Episodic memory was related to ventral prefrontal cortex-thalamo-hippocampal fiber integrity; (b) Working memory was related to integrity of corpus callosum body fibers subserving dorsolateral prefrontal cortex; and (c) Reasoning was related to integrity of corpus callosum body fibers subserving rostral and caudal dorsolateral prefrontal cortex. These findings confirm the ventrolateral prefrontal cortex''s role in semantic control and the dorsolateral prefrontal cortex''s role in rule-based processing, in accordance with the dorso-ventral prefrontal cortex gradient. Reasoning-related rostral and caudal superior frontal white matter may facilitate different levels of task rule complexity. This study is the first to demonstrate dorso-ventral and rostro-caudal prefrontal cortex processing gradients in white matter integrity.