Gastrula Ectoderm Research Articles

Temporal dynamics of BMP/Nodal ratio drive tissue-specific gastrulation morphogenesis.

Anteroposterior (AP) elongation of the vertebrate body plan is driven by convergence and extension (C&E) gastrulation movements in both the mesoderm and neuroectoderm, but how or whether molecular regulation of C&E differs between tissues remains an open question. Using a zebrafish explant model of AP axis extension, we show that C&E of the neuroectoderm and mesoderm can be uncoupled ex vivo, and that morphogenesis of individual tissues results from distinct morphogen signaling dynamics. Using precise temporal manipulation of BMP and Nodal signaling, we identify a critical developmental window during which high or low BMP/Nodal ratios induce neuroectoderm- or mesoderm-driven C&E, respectively. Increased BMP activity similarly enhances C&E specifically in the ectoderm of intact zebrafish gastrulae, highlighting the in vivo relevance of our findings. Together, these results demonstrate that temporal dynamics of BMP and Nodal morphogen signaling activate distinct morphogenetic programs governing C&E gastrulation movements within individual tissues.

FGF/MAPK/Ets signaling in Xenopus ectoderm contributes to neural induction and patterning in an autonomous and paracrine manner, respectively.

FGF and anti-BMP signals from the Spemann organizer of mesodermal origin are essential for Xenopus neural development from gastrula ectoderm. However, the detailed cellular and molecular mechanisms of signaling, especially those underlying the neural induction process, are still controversial. We show here that the expression of early neural marker genes such as sox2 and otx2 is suppressed both in vivo and in vitro, when ectoderm cells are loaded with a dominant-negative construct of Ets transcription factors or a translation-blocking antisense FGF2 MO or FGF8 MO, respectively. This indicates that the expression of these FGF signaling molecules in ectoderm cells contributes significantly to neural induction, in contrast to the "neural default model" that has been emphasized, in which anti-BMP signaling from the organizer plays a major role in the neural induction in Xenopus. Our results indicate that cell-autonomous FGF signaling between ectoderm cells, rather than paracrine signaling from organizer cells, directly induces the expression of sox2 and otx2 via the FGF2, FGF8/MAPK/Ets pathway at very low signaling levels. This is independent of inhibiting BMP signaling via the FGF/MAPK/Smad pathway, which has been proposed as the primary pathway for FGF signaling in Xenopus neural induction. Using cultured ectoderm cells, we also obtained results suggesting that the mode of contribution of FGF signaling to neural development is altered during the subsequent neural patterning stage. As we increase the amounts of FGF in the culture medium, anterior neural genes activated at low FGF signaling levels are suppressed, and instead position-specific, more posterior neural genes are activated in a dose-dependent manner via the FGF/MAPK/Ets pathway. These results support the claim that morphogenic FGFs from the organizer diffuse in a paracrine manner through the plane of the induced neuroectoderm, causing anteroposterior patterning of neural tissue.

HES and Mox genes are expressed during early mesoderm formation in a mollusk with putative ancestral features

The mesoderm is considered the youngest of the three germ layers. Although its morphogenesis has been studied in some metazoans, the molecular components underlying this process remain obscure for numerous phyla including the highly diverse Mollusca. Here, expression of Hairy and enhancer of split (HES), Mox, and myosin heavy chain (MHC) was investigated in Acanthochitona fascicularis, a representative of Polyplacophora with putative ancestral molluscan features. While AfaMHC is expressed throughout myogenesis, AfaMox1 is only expressed during early stages of mesodermal band formation and in the ventrolateral muscle, an autapomorphy of the polyplacophoran trochophore. Comparing our findings to previously published data across Metazoa reveals Mox expression in the mesoderm in numerous bilaterians including gastropods, polychaetes, and brachiopods. It is also involved in myogenesis in molluscs, annelids, tunicates, and craniates, suggesting a dual role of Mox in mesoderm and muscle formation in the last common bilaterian ancestor. AfaHESC2 is expressed in the ectoderm of the polyplacophoran gastrula and later in the mesodermal bands and in putative neural tissue, whereas AfaHESC7 is expressed in the trochoblasts of the gastrula and during foregut formation. This confirms the high developmental variability of HES gene expression and demonstrates that Mox and HES genes are pleiotropic.

An essential role for LPA signalling in telencephalon development

Lysophosphatidic acid (LPA) has wide-ranging effects on many different cell types, acting through G-protein-coupled receptors such as LPAR6. We show that Xenopus lpar6 is expressed from late blastulae and is enriched in the mesoderm and dorsal ectoderm of early gastrulae. Expression in gastrulae is an early response to FGF signalling. Transcripts for lpar6 are enriched in the neural plate of Xenopus neurulae and loss of function caused forebrain defects, with reduced expression of telencephalic markers (foxg1, emx1 and nkx2-1). Midbrain (en2) and hindbrain (egr2) markers were unaffected. Foxg1 expression requires LPAR6 within ectoderm and not mesoderm. Head defects caused by LPAR6 loss of function were enhanced by co-inhibiting FGF signalling, with defects extending into the hindbrain (en2 and egr2 expression reduced). This is more severe than expected from simple summation of individual defects, suggesting that LPAR6 and FGF have overlapping or partially redundant functions in the anterior neural plate. We observed similar defects in forebrain development in loss-of-function experiments for ENPP2, an enzyme involved in the synthesis of extracellular LPA. Our study demonstrates a role for LPA in early forebrain development.

Novel animal pole‐enriched maternal mRNAs are preferentially expressed in neural ectoderm

Many animals utilize maternal mRNAs to pre-pattern the embryo before the onset of zygotic transcription. In Xenopus laevis, vegetal factors specify the germ line, endoderm, and dorsal axis, but there are few studies demonstrating roles for animal-enriched maternal mRNAs. Therefore, we carried out a microarray analysis to identify novel maternal transcripts enriched in 8-cell-stage animal blastomeres. We identified 39 mRNAs isolated from 8-cell animal blastomeres that are >4-fold enriched compared to vegetal pole mRNAs. We characterized 14 of these that are of unknown function. We validated the microarray results for 8/14 genes by qRT-PCR and for 14/14 genes by in situ hybridization assays. Because no developmental functions are reported yet, we provide the expression patterns for each of the 14 genes. Each is expressed in the animal hemisphere of unfertilized eggs, 8-cell animal blastomeres, and diffusely in blastula animal cap ectoderm, gastrula ectoderm and neural ectoderm, neural crest (and derivatives) and cranial placodes (and derivatives). They have varying later expression in some mesodermal and endodermal tissues in tail bud through larval stages. Novel animal-enriched maternal mRNAs are preferentially expressed in ectodermal derivatives, particularly neural ectoderm. However, they are later expressed in derivatives of other germ layers.

Waterborne exposure to clodinafop‐propargyl disrupts the posterior and ventral development of zebrafish embryos

Clodinafop-propargyl, an aryloxyphenoxypropionate herbicide, is widely used for the control of annual grasses. However, research focusing on the ecotoxicity of this herbicide is limited. The present study employed zebrafish (Danio rerio) as a model to investigate its developmental toxicity. Embryos were exposed to a range of concentrations from 0.2 µM to 5 µM starting at late cleavage stage (2 h postfertilization, [hpf]) or late gastrulation stage (10 hpf). The results showed that the two exposure strategies had the same minimum teratogenic concentration of 0.6 µM but caused different groups of morphogenetic malformations. When exposure was initiated at 2 hpf, clodinafop-propargyl caused various embryonic phenotypes, including embryos with a fin gap in the ventral tail and embryos with coiled tail. When exposure was initiated at 10 hpf, clodinafop-propargyl resulted in failure of the tail to detach, in which the ventral tissues failed to grow out but instead adhered to the yolk extension, and the defect differed to various degrees among embryos. Similar effects were observed for embryos exposed to clodinafop, the metabolite of clodinafop-propargyl. Because these defects were mainly confined to the posterior and ventral region that derived from ventral blastoderm cells, we have evaluated the expression of the ventral mesoderm marker gene gata-1 and ventral ectoderm marker gene gata-3. No significant alteration was seen in gata-1 expression except for the expanded blood islands, whereas the expression of gata-3 was significantly reduced. Our findings showed that clodinafop-propargyl exposure disturbed embryonic patterning and fate specification of ventrally derived gastrula ectoderm cells.

Induction and patterning of trunk and tail neural ectoderm by the homeobox gene eve1 in zebrafish embryos

In vertebrates, Evx homeodomain transcription factor-encoding genes are expressed in the posterior region during embryonic development, and overexpression experiments have revealed roles in tail development in fish and frogs. We analyzed the molecular mechanisms of posterior neural development and axis formation regulated by eve1. We show that eve1 is involved in establishing trunk and tail neural ectoderm by two independent mechanisms: First, eve1 posteriorizes neural ectoderm via induction of aldh1a2, which encodes an enzyme that synthesizes retinoic acid; second, eve1 is involved in neural induction in the posterior ectoderm by attenuating BMP expression. Further, eve1 can restore trunk neural tube formation in the organizer-deficient ichabod(-/-) mutant. We conclude that eve1 is crucial for the organization of the antero-posterior and dorso-ventral axis in the gastrula ectoderm and also has trunk- and tail-promoting activity.

Osteopathy and (hatha) yoga

Differences and points of contact between osteopathy and yoga as regards their history and practical application are outlined. Both seek to promote healing. Yoga seeks the attainment of consciousness; osteopathy aims for providing support to health. One fundamental difference is the personal involvement of the individual in yoga. Teacher and student alike are challenged to re-examine the attitudes of mind they have adopted toward their lives. Osteopathy generally involves a relatively passive patient while the osteopath is active in providing treatment. Practical examples are used to highlight points of contact between yoga and osteopathy. The text includes a discussion of the importance of physicality and a description of ways of using it in healing processes. Furthermore, processes of attaining consciousness are outlined. Possible reductionist misconceptions in yoga and osteopathy are also pointed out. Fundamental attitudes and focus that complement each other are presented, taking the concept of stillness as a particular example.

Ernest Everett Just, Johannes Holtfreter, and the origin of certain concepts in embryo morphogenesis.

Ernest E. Just (1883-1941) is best known for his discovery of the "wave of negativity" that sweeps of the sea urchin egg during fertilization, and his elucidation of what are known as the fast and slow blocks to polyspermy. Just's contemporary Johannes Holtfreter (1901-1992) is known for his pioneering work in amphibian morphogenesis, which helped to lay the foundation for modern vertebrate developmental biology. This paper, after briefly describing the life and scientific contributions of Just, argues that his work and ideas strongly influenced two of the concepts for which Holtfreter is best known: tissue affinity and autoneuralization (or autoinduction). Specifically, this paper argues that, first, Just's experiments demonstrating developmental stage-specific changes in the adhesiveness of the blastomeres of cleavage embryos helped lay the foundation for Holtfreter's concept of tissue affinity and, second, Just's notion of the intrinsic irritability of the egg cell, which is evident in experimental parthenogenesis, strongly informed Holtfreter's concept of the nonspecific induction of neural tissue formation in amphibian gastrula ectoderm explants, a phenomenon known as autoinduction. Acknowledgment of these contributions by Just in no way diminishes the importance of Holtfreter's groundbreaking work. It does, however, extend the impact of Just's work into the area of embryo morphogenesis. It connects Just to Holtfreter and positions his work as an antecedent to embryo research that continues to this day.

Ontogeny of the holothurian larval nervous system: evolution of larval forms

Echinoderm larvae share numerous features of neuroanatomy. However, there are substantial differences in specific aspects of neural structure and ontogeny between the dipleurula-like larvae of asteroids and the pluteus larvae of echinoids. To help identify apomorphic features, we have examined the ontogeny of the dipleurula-like auricularia larva of the sea cucumber, Holothuria atra. Neural precursors arise in the apical ectoderm of gastrulae and appear to originate in bilateral clusters of cells. The cells differentiate without extensive migration, and they align with the developing ciliary bands and begin neurogenesis. Neurites project along the ciliary bands and do not appear to extend beneath either the oral or aboral epidermis. Apical serotonergic cells are associated with the preoral loops of the ciliary bands and do not form a substantial commissure. Paired, tripartite connectives form on either side of the larval mouth that connect the pre-oral, post-oral, and lateral ciliary bands. Holothurian larvae share with hemichordates and bipinnariae a similar organization of the apical organ, suggesting that the more highly structured apical organ of the pluteus is a derived feature. However, the auricularia larva shares with the pluteus larva of echinoids several features of neural ontogeny. Both have a bilateral origin of neural precursors in ectoderm adjacent to presumptive ciliary bands, and the presumptive neurons move only a few cell diameters before undergoing neurogenesis. The development of the holothurian nervous systems suggests that the extensive migration of neural precursors in asteroids is a derived feature.

Grainyhead-like 3, a transcription factor identified in a microarray screen, promotes the specification of the superficial layer of the embryonic epidermis

The Xenopus ectoderm consists of two populations of cells, superficial polarised epithelial cells and deep, non-epithelial cells. These two cell types differ in their developmental fate. In the neural ectoderm, primary neurons are derived only from the deep cells. In the epidermal ectoderm, superficial cells express high levels of differentiation markers, while most of the deep cells do not differentiate until later when they produce the stratified adult epidermis. However, few molecular differences are known between the deep and superficial cells. Here, we have undertaken a systematic approach to identify genes that show layer-restricted expression by microarray analysis of deep and superficial cells at the gastrula stage, followed by wholemount in situ hybridisation. We have identified 32 differentially expressed genes, of which 26 show higher expression in the superficial layer and 6 in the deep layer and describe their expression at the gastrula and neurula stage. One of the identified genes is the transcription factor Grhl3, which we found to be expressed in the superficial layer of the gastrula ectoderm and the neurula epidermis. By using markers identified in this work, we show that Grlh3 promotes superficial gene expression in the deep layer of the epidermis. Concomitantly, deep layer specific genes are switched off, showing that Grlh3 can promote deep cells to take on a superficial cell identity in the embryonic epidermis.

Combinatorial Fgf and Bmp signalling patterns the gastrula ectoderm into prospective neural and epidermal domains.

Studies in fish and amphibia have shown that graded Bmp signalling activity regulates dorsal-to-ventral (DV) patterning of the gastrula embryo. In the ectoderm, it is thought that high levels of Bmp activity promote epidermal development ventrally, whereas secreted Bmp antagonists emanating from the organiser induce neural tissue dorsally. However, in zebrafish embryos, the domain of cells destined to contribute to the spinal cord extends all the way to the ventral side of the gastrula, a long way from the organiser. We show that in vegetal (trunk and tail) regions of the zebrafish gastrula, neural specification is initiated at all DV positions of the ectoderm in a manner that is unaffected by levels of Bmp activity and independent of organiser-derived signals. Instead, we find that Fgf activity is required to induce vegetal prospective neural markers and can do so without suppressing Bmp activity. We further show that Bmp signalling does occur within the vegetal prospective neural domain and that Bmp activity promotes the adoption of caudal fate by this tissue.

Analysis of Wnt8 for neural posteriorizing factor by identifying Frizzled 8c and Frizzled 9 as functional receptors for Wnt8

The dorsal ectoderm of vertebrate gastrula is first specified into anterior fate by an activation signal and posteriorized by a graded transforming signal, leading to the formation of forebrain, midbrain, hindbrain and spinal cord along the anteroposterior (A–P) axis. Transplanted non-axial mesoderm rather than axial mesoderm has an ability to transform prospective anterior neural tissue into more posterior fates in zebrafish. Wnt8 is a secreted factor that is expressed in non-axial mesoderm. To investigate whether Wnt8 is the neural posteriorizing factor that acts upon neuroectoderm, we first assigned Frizzled 8c and Frizzled 9 to be functional receptors for Wnt8. We then, transplanted non-axial mesoderm into the embryos in which Wnt8 signaling is cell-autonomously blocked by the dominant-negative form of Wnt8 receptors. Non-axial mesodermal transplants in embryos in which Wnt8 signaling is cell-autonomously blocked induced the posterior neural markers as efficiently as in wild-type embryos, suggesting that Wnt8 signaling is not required in neuroectoderm for posteriorization by non-axial mesoderm. Furthermore, Wnt8 signaling, detected by nuclear localization of β-catenin, was not activated in the posterior neuroectoderm but confined in marginal non-axial mesoderm. Finally, ubiquitous over-expression of Wnt8 does not expand neural ectoderm of posterior character in the absence of mesoderm or Nodal-dependent co-factors. We thus conclude that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A–P axis.

A Quantitative Study of Regional and Stage Specific Reaction of Xenopus laevis Embryonic Tissues on Mechanical Load

Residual deformation of fragments of the embryonic tissues preserved after relaxation of the stretching force serve as a criterion of active redistribution of their cells caused by this stretching. We measured residual deformations of the Xenopus laevis ventral and dorsal ectoderm at the early gastrula and lateral ectoderm at the late gastrula-early neurula after stretching of varying time and force. While the samples responded to moderate (up to 40%) short-term stretching as elastic bodies (residual deformations were absent), residual deformation appeared in the early gastrula tissues after 30–60-min stretching, which were more pronounced in the ventral tissues than in the dorsal ones. On the contrary, a contractile reaction developed in the late gastrula-early neurula tissues in response to 60-min stretching, which almost relaxed residual deformation within 20 min after unloading. A conclusion was drawn that gastrulation and neurulation proceed under the conditions of relaxing and nonrelaxing mechanical tensions, respectively. Mechanical bases and morphogenetic role of the described reactions is discussed.

Gene profiling during neural induction in Xenopus laevis: regulation of BMP signaling by post-transcriptional mechanisms and TAB3, a novel TAK1-binding protein.

The earliest decision in vertebrate neural development is the acquisition of a neural identity by embryonic ectodermal cells. The default model for neural induction postulates that neural fate specification in the vertebrate embryo occurs by inhibition of epidermal inducing signals in the gastrula ectoderm. Bone morphogenetic proteins (BMPs) act as epidermal inducers, and all identified direct neural inducers block BMP signaling either intra- or extracellularly. Although the mechanism of action of the secreted neural inducers has been elucidated, the relevance of intracellular BMP inhibitors in neural induction is not clear. In order to address this issue and to identify downstream targets after BMP inhibition, we have monitored the transcriptional changes in ectodermal explants neuralized by Smad7 using a Xenopus laevis 5000-clone gastrula-stage cDNA microarray. We report the identification and initial characterization of 142 genes whose transcriptional profiles change in the neuralized explants. In order to address the potential involvement during neural induction of genes identified in the array, we performed gain-of-function studies in ectodermal explants. This approach lead to the identification of four genes that can function as neural inducers in Xenopus and three others that can synergize with known neural inducers in promoting neural fates. Based on these studies, we propose a role for post-transcriptional control of gene expression during neural induction in vertebrates and present a model whereby sustained BMP inhibition is promoted partly through the regulation of TGFbeta activated kinase (TAK1) activity by a novel TAK1-binding protein (TAB3).

Nuclear exclusion of Smad2 is a mechanism leading to loss of competence.

Controlling the duration of a signalling process in development by loss of competence is important because too strong an induction can change cell fate. To understand some of the mechanisms that underlie loss of competence, we have analysed the transduction of transforming growth factor-beta (TGF-beta) signalling during mesoderm formation, which is thought to be induced by TGF-beta-like signalling, in embryos of the frog Xenopus laevis. Here we show that gastrula ectoderm has the ability to express mesodermal marker genes in response to the TGF-beta signalling molecule activin for many hours, but then loses this ability within 1 h for all mesodermal genes tested. This loss of mesodermal competence correlates with the inability of Smad2, the principal intracellular signal transducer of activin, to accumulate in the nucleus. Mutating three phosphorylation sites within Smad2 abrogates the temporal restriction of Smad2 to accumulate in the nucleus. Overexpression of this mutant form of Smad2 can prolong the competence of endogenous mesodermal genes to respond to activin signalling. Thus, restricting the subcellular localization of an intracellular signal transducer constitutes a mechanism that leads to loss of mesodermal competence. This mechanism operates within less than an hour, and is therefore well suited to control an orderly sequence of inductions.

Inductive activity of retinal peptides.

We studied the effect of retinal polypeptide Retinalamin on multipotent ectodermal cells of the early gastrula of Xenopus laevis. Neuronal differentiation of the early gastrula ectoderm including the brain, retina, and pigment epithelium depended on Retinalamin concentration.

Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo.

The dorsal ectoderm of the vertebrate gastrula was proposed by Nieuwkoop to be specified towards an anterior neural fate by an activation signal, with its subsequent regionalization along the anteroposterior (AP) axis regulated by a graded transforming activity, leading to a properly patterned forebrain, midbrain, hindbrain and spinal cord. The activation phase involves inhibition of BMP signals by dorsal antagonists, but the later caudalization process is much more poorly characterized. Explant and overexpression studies in chick, Xenopus, mouse and zebrafish implicate lateral/paraxial mesoderm in supplying the transforming influence, which is largely speculated to be a Wnt family member. We have analyzed the requirement for the specific ventrolaterally expressed Wnt8 ligand in the posteriorization of neural tissue in zebrafish wild-type and Nodal-deficient embryos (Antivin overexpressing or cyclops;squint double mutants), which show extensive AP brain patterning in the absence of dorsal mesoderm. In different genetic situations that vary the extent of mesodermal precursor formation, the presence of lateral wnt8-expressing cells correlates with the establishment of AP brain pattern. Cell tracing experiments show that the neuroectoderm of Nodal-deficient embryos undergoes a rapid anterior-to-posterior transformation in vivo during a short period at the end of the gastrula stage. Moreover, in both wild-type and Nodal-deficient embryos, inactivation of Wnt8 function by morpholino (MO(wnt8)) translational interference dose-dependently abrogates formation of spinal cord and posterior brain fates, without blocking ventrolateral mesoderm formation. MO(wnt8) also suppresses the forebrain deficiency in bozozok mutants, in which inactivation of a homeobox gene causes ectopic wnt8 expression. In addition, the bozozok forebrain reduction is suppressed in bozozok;squint;cyclops triple mutants, and is associated with reduced wnt8 expression, as seen in cyclops;squint mutants. Hence, whereas boz and Nodal signaling largely cooperate in gastrula organizer formation, they have opposing roles in regulating wnt8 expression and forebrain specification. Our findings provide strong support for a model of neural transformation in which a planar gastrula-stage Wnt8 signal, promoted by Nodal signaling and dorsally limited by Bozozok, acts on anterior neuroectoderm from the lateral mesoderm to produce the AP regional patterning of the CNS.

Regulation of MAP Kinase by the BMP-4/TAK1 Pathway in Xenopus Ectoderm

Bone morphogenetic protein-4 (BMP-4) induces epidermis and represses neural fate in Xenopus ectoderm. Our previous findings implicate p42 Erk MAP kinase (MAPK) in the response to neural induction. We have examined the effects of BMP-4 on MAPK activity in gastrula ectoderm. Expression of a dominant negative BMP-4 receptor resulted in a 4.5-fold elevation in MAPK activity in midgastrula ectoderm. MAPK activity was reduced in ectoderm expressing a constitutively active BMP-4 receptor, or ectoderm treated with BMP-4 protein in the presence or absence of cycloheximide. Overexpression of TAK1 led to a reduction in MAPK activity in early gastrula ectoderm. The inhibitory effects of TAK1 could be reversed by 1 μM SB 203580, a p38 inhibitor. Treatment of isolated ectoderm with SB 203580 led to expression of otx2, NCAM, and noggin. Western blot analyses indicated that the BMP-4 pathway does not activate JNKs in ectoderm. Our findings indicate that BMP-4 inhibits ectodermal MAPK activity through a TAK1/p38-type pathway. MAPK has been shown to inactivate Smad1. Thus, our results suggest that BMP-4 and MAPK pathways are mutually antagonistic in Xenopus ectoderm, and that interactions between these pathways may govern the choice between epidermal and neural fate.

Differential regulation of Dlx gene expression by a BMP morphogenetic gradient.

Three members of the vertebrate Distal-less gene family, Dlx3, 5 and 6, are transcribed in early gastrula embryos of Xenopus laevis. This expression is confined to ectoderm and is excluded from the presumptive neural plate region. Expression of all three genes is dependent upon BMP signaling, with significant differences in how the three genes respond to the BMP antagonist chordin. This correlates with the different expression domain boundaries in vivo for Dlx3 compared to Dlx5 and 6, suggesting that BMP signal attenuation could be the primary factor in determining these different patterns in the gastrula ectoderm.