Phenoloxidase, a marker enzyme for differentiation of the neural ectoderm and the epidermal ectoderm during embryonic development of amphioxus Branchiostoma belcheri tsingtaunese

The development of phenoloxidase during amphioxus embryogenesis was spectrophotometrically and histochemically studied for the first time in the present study. It was found that (1) PO activity initially appeared in the general ectoderm including the neural ectoderm and the epidermal ectoderm at the early neurula stage but not in the mesoderm or the endoderm, and (2) PO activity disappeared in the neural plate cells but remained unchanged in the epidermal cells when the neural plate was morphologically quite distinct from the rest of the ectoderm. It is apparent that PO could serve as a marker enzyme for differentiation of the neural ectoderm from the epidermal ectoderm during embryonic development of amphioxus.     

Retinoic-acid signalling in node ectoderm and posterior neural plate directs left-right patterning of somitic mesoderm

Somitogenesis requires bilateral rhythmic segmentation of paraxial mesoderm along the antero-posterior axis. The location of somite segmentation depends on opposing signalling gradients of retinoic acid (generated by retinaldehyde dehydrogenase-2; Raldh2) anteriorly and fibroblast growth factor (FGF; generated by Fgf8) posteriorly. Retinoic-acid-deficient embryos exhibit somite left-right asymmetry, but it remains unclear how retinoic acid mediates left-right patterning. Here, we demonstrate that retinoic-acid signalling is uniform across the left-right axis and occurs in node ectoderm but not node mesoderm. In Raldh2(-/-) mouse embryos, ectodermal Fgf8 expression encroaches anteriorly into node ectoderm and neural plate, but its expression in presomitic mesoderm is initially unchanged. The late stages of somitogenesis were rescued in Raldh2(-/-) mouse embryos when the maternal diet was supplemented with retinoic acid until only the 6-somite stage, demonstrating that retinoic acid is only needed during node stages. A retinoic-acid-reporter transgene marking the action of maternal retinoic acid in rescued Raldh2(-/-) embryos revealed that the targets of retinoic-acid signalling during somitogenesis are the node ectoderm and the posterior neural plate, not the presomitic mesoderm. Our findings suggest that antagonism of Fgf8 expression by retinoic acid occurs in the ectoderm and that failure of this mechanism generates excessive FGF8 signalling to adjacent mesoderm, resulting initially in smaller somites and then left-right asymmetry.     

Coelom formation: binary decision of the lateral plate mesoderm is controlled by the ectoderm.

Most triploblastic animals including vertebrates have a coelomic cavity that separates the outer and inner components of the body. The coelom is lined by two different tissue components, somatopleure and splanchnopleure, which are derived from the lateral plate region. Thus, the coelom is constructed as a result of a binary decision during early specification of the lateral plate. In this report we studied the molecular mechanisms of this binary decision. We first demonstrate that the splitting of the lateral plate into the two cell sheets progresses in an anteroposterior order and this progression is not coordinated with that of the somitic segmentation. By a series of embryological manipulations we found that young splanchnic mesoderm is still competent to be respecified as somatic mesoderm, and the ectoderm overlying the lateral plate is sufficient for this redirection. The lateral ectoderm is also required for maintenance of the somatic character of the mesoderm. Thus, the ectoderm plays at least two roles in the early subdivision of the lateral plate: specification and maintenance of the somatic mesoderm. We also show that the latter interactions are mediated by BMP molecules that are localized in the lateral ectoderm. Evolutionary aspects of the coelom formation are also considered.     

Homeogenetic neural induction in Xenopus

Neural induction is known to involve an interaction of ectoderm with dorsal mesoderm during gastrulation, but several kinds of studies have argued that competent ectoderm can also be neutralized via an interaction with previously neuralized tissue, a process termed homeogenetic neural induction. Although homeogenetic neural induction has been proposed to play an important role in the normal induction of neural tissue, this process has not been subjected to detailed study using tissue recombinants and molecular markers. We have examined the question of homeogenetic neural induction in Xenopus embryos, both in transplant and recombinant experiments, using the expression of two neural antigens to assay the response. When ectoderm that is competent to be neuralized is transplanted to the region adjacent to the neural plate of early neurula embryos, it forms neural tissue, as assayed by staining with antibodies against the neural cell adhesion molecule, N-CAM. Transplants to the ventral region, far from the neural plate, do not express N-CAM, indicating that neuralization is not occurring as a result of the transplantation procedure itself. Because this response might be occurring as a result of interactions of ectoderm with either adjacent neural plate tissue, or with underlying dorsolateral mesoderm, recombinant experiments were performed to determine the source of the neuralizing signal. Ectoderm cultured in combination with neural plate tissue alone expresses neural markers, while ectoderm cultured in combination with dorsolateral mesoderm does not. We conclude that neural tissue can homeogenetically induce competent ectoderm to form neural tissue and argue that this induction occurs via planar signaling within the ectoderm, a mechanism that, in normal development, may be involved in interactions within presumptive neural ectoderm or in specifying structures that lie near the neural plate.     

A labile period in the determination of the anterior-posterior axis during early neural development in Xenopus.

The process by which the vertebrate central nervous system acquires its regional properties remains a central problem in developmental biology. It is generally argued that at early gastrula stages the dorsal mesoderm possesses precise anterior-posterior positional information, which is subsequently imparted to the overlying ectoderm. However, using regionally specific gene probes to monitor regional responses in Xenopus embryos, we find that anterior-posterior properties are not fixed until early neurula stages. During gastrulation the regional inducing capacities of the dorsal mesoderm as well as the regional responses of the presumptive neural ectoderm are activated along the entire anterior-posterior axis when these properties are assayed in recombinant and explant experiments, respectively. Restriction of regional inducing capacity in the mesoderm and responsiveness in the neural ectoderm occur only at neural plate stages.     

A novel homeobox gene expressed in the anterior neural plate of the Xenopus embryo.

To obtain gene sequences controlling the early steps of amphibian neurogenesis, we have performed differential screening of a subtractive cDNA library prepared by a novel PCR-based method from a single presumptive neural plate of a Xenopus laevis late-gastrula embryo. As a result we have isolated a fragment of a novel homeobox gene (named XANF-1, for Xenopus anterior neural folds). This gene is expressed predominantly in the anterior part of the developing nervous system. Such preferential localization of XANF-1 mRNA is established from its initially homogenous distribution in ectoderm of early gastrula. This change in the expression pattern is conditioned by a differential influence of various mesoderm regions on ectoderm: anterior mesoderm activates XANF-1 expression in the overlying ectoderm, whereas posterior axial and ventral mesoderm areas inhibit it. The data obtained demonstrate for the first time that selection of genes for specific expression in the CNS of the early vertebrate embryo is affected not only by chordamesoderm (a neural inductor) but also by ventral mesoderm.     

The activation of a neuralizing factor in the neural plate is correlated with its homoiogenetic-inducing activity

Summary Neural plates which are induced in the dorsal ectoderm of Triturus by the underlying mesoderm acquire, in turn, neural-inducing activity. This process is correlated with the appearance of neural-inducing activity in the microsomal fraction of the neural plate homogenate. The high-speed supernatant also acquires inducing activity after neural induction, but to a lesser extent. The experiments suggest that a masked neuralizing factor, which is already present in the ectoderm, is in part activated and exported from the inducing neural plate cells.     

Progressive determination during formation of the anteroposterior axis in Xenopus laevis

The cement gland is an ectodermal organ in the head of frog embryos, lying anterior to any neural tissue. As analyzed by specific RNA expression, cement gland, like neural tissue, was induced by the dorsal mesoderm. Interestingly, mesoderm with the highest cement gland-inducing potential lay posterior to the ectoderm fated to form this organ, indicating that its induction occurred at a distance from the inducer source. Cement gland induction first occurred during early gastrulation. However, most initially induced cells did not contribute to the mature cement gland, but instead formed part of the neural plate. This change in fate could be reconstituted in vitro. These results suggest that determination of part of the anteroposterior axis occurs progressively, where future neural ectoderm is first induced to a cement glandlike state. As gastrulation proceeds, further induction by mesoderm may override this state, which persists only in the extreme anterior of the embryo.     

Signals derived from the underlying mesoderm are dispensable for zebrafish neural crest induction

Signals from the non-neural ectoderm, the neural ectoderm, and the underlying mesoderm have all been implicated in the induction of neural crest. Bone morphogenetic protein (BMP) signaling in particular has an important role in this process; however, it is unclear whether this activity of BMP is due to its effects on patterning the underlying mesoderm, to its ability to establish a competent neural plate boundary zone, or to the direct specification of neural crest at intermediate levels of activity within a BMP gradient. We show neural crest induction occurs in zebrafish in the absence of involuted mesoderm, indicating that this tissue and signals derived from it are dispensable for the formation of neural crest. Dorsal-involuted mesoderm is a major source of secreted BMP antagonists, and the activity of BMP signaling is thought to depend on the presence of the opposing activity of these antagonists. We find that the three BMP antagonists known to be expressed during gastrulation in zebrafish, noggin1, follistatin, and chordin, are dispensable for neural crest induction. These results suggest that mechanisms for restricting the spatio-temporal pattern of BMP expression may compensate for the loss of secreted BMP antagonist activity in establishing dorso-ventral patterning, neural induction, and the neural crest.     

Mesoderm induction in the future tail region of Xenopus

Summary We have used interspecific grafts between Xenopus borealis and Xenopus laevis to study the signalling system that produces tail mesoderm. Early gastrula ectoderm grafted into the posterior neural plate region of neurulae responds to a mesodermal inducing signal in this region and forms mainly tail somites; this signal persists until at least the early tail bud stage. Ventral ectoderm grafted into the posterior neural plate loses its competence to respond to this signal after stage 10 1/2. We have established the specification of anterior and posterior neural plate ectoderm. In ectodermal sandwiches or when grafted into unusual positions, anterior regions gave rise to mainly nervous system and posterior regions to large amounts of muscle, together with some nervous system. Thus it was impossible to assess the competence of posterior neural plate ectoderm to form further mesoderm and hence to establish if mesodermal induction continues during neurulation in unmanipulated embryos.     

Surface ectoderm is necessary for the morphogenesis of somites

The paraxial mesoderm of the neck and trunk of mouse embryos undergoes extensive morphogenesis in forming somites. Paraxial mesoderm is divided into segments, it elongates along its anterior posterior axis, and its cells organize into epithelia. Experiments were performed to determine if these processes are autonomous to the mesoderm that gives rise to the somites. Presomitic mesoderm at the tailbud stage was cultured in the presence and absence of its adjacent tissues. Somite segmentation occurred in the absence of neural tube, notochord, gut and surface ectoderm, and occurred in posterior fragments in the absence of anterior presomitic mesoderm. Mesodermal expression of Dll1 and Notch1, genes with roles in segmentation, was largely independent of other tissues, consistent with autonomous segmentation. However, surface ectoderm was found to be necessary for elongation of the mesoderm along the anterior-posterior axis and for somite epithelialization. To determine if there is specificity in the interaction between ectoderm and mesoderm, ectoderm from different sources was recombined with presomitic mesoderm. Surface ectoderm from only certain parts of the embryo supported somite epithelialization and elongation. Somite epithelialization induced by ectoderm was correlated with expression of the basic-helix-loop-helix gene Paraxis in the mesoderm. This is consistent with the genetically defined requirement for Paraxis in somite epithelialization. However, trunk ectoderm was able to induce somite epithelialization in the absence of strong Paraxis expression. We conclude that somitogenesis consists of autonomous segmentation patterned by Notch signaling and nonautonomous induction of elongation and epithelialization by surface ectoderm.     

Neural induction in embryos

Neural differentiation of the ectoderm is inhibited by bone morphogenetic protein 4 (BMP-4) in amphibia as well as mammalia. This inhibition is released by neural inducing factor(s), which are secreted from the dorsal mesoderm. Masked neuralizing factor(s) are already present in the ectoderm before induction. In homogenates from Xenopus oocytes and embryos neural inducing factors were found in the supernatant (centrifuged at 105 000 g ), in small vesicles and a ribonucleoprotein fraction. A neuralizing factor, which is a protein of small size, has been partially purified from Xenopus gastrulae. Genes that are expressed in the dorsal mesoderm and involved in the de novo synthesis of neuralizing factor(s) have been cloned. The differentiation of cells with a neuronal fate starts in the neural plate immediately after neural induction. Genes homologous to the Notch and Delta genes of lateral inhibition in insects are involved in this process.     

Regulation of ectodermal Wnt6 expression by the neural tube is transduced by dermomyotomal Wnt11: a mechanism of dermomyotomal lip sustainment

Ectodermal Wnt6 plays an important role during development of the somites and the lateral plate mesoderm. In the course of development, Wnt6 expression shows a dynamic pattern. At the level of the segmental plate and the epithelial somites, Wnt6 is expressed in the entire ectoderm overlying the neural tube, the paraxial mesoderm and the lateral plate mesoderm. With somite maturation, expression becomes restricted to the lateral ectoderm covering the ventrolateral lip of the dermomyotome and the lateral plate mesoderm. To study the regulation of Wnt6 expression, we have interfered with neighboring signaling pathways. We show that Wnt1 and Wnt3a signaling from the neural tube inhibit Wnt6 expression in the medial surface ectoderm via dermomyotomal Wnt11. We demonstrate that Wnt11 is an epithelialization factor acting on the medial dermomyotome, and present a model suggesting Wnt11 and Wnt6 as factors maintaining the epithelial nature of the dorsomedial and ventrolateral lips of the dermomyotome, respectively, during dermomyotomal growth.     

Morphogenesis of the cranial segments and distribution of neural crest in the embryos of the snapping turtle,Chelydra serpentina

Recent studies of the heads of vertebrates have shown a primitive pattern of segmentation in the mesoderm and neural plate not previously recognized. The role of this pattern in the subsequent distribution of cranial crest and the development of branchial arches and cranial nerves, may resolve century-old arguments about the evolution of vertebrate segmentation. In this study, we examine the early embryonic development of the cranium of a primitive amniote, the snapping turtle, with the SEM. We show that the paraxial mesoderm cranial to the first-formed somites is segmented and that this pattern is based on somitomeres, similar to those described in the embryos of chick and mouse. Seven contiguous pairs of somitomeres comprise the “head mesoderm”; the first pair of somites actually arise from the eighth pair of somitomeres added to the axis. Cranial somitomeres are associated with specific brain regions, in that the first pair lie adjacent to prosencephalon, the second and third pair are adjacent to the mesencephalon, and the fourth, fifth, sixth, and seventh pair of somitomeres lie adjacent to individual neuromeres of the rhombencephalon. Prior to the closure of the anterior neuropore, cranial neural crest cells first emerge from the mesencephalon and migrate onto the second and third somitomeres. Shortly thereafter, neural crest cells emerge at more caudal levels of the rhombencephalon, beginning at the juncture of the fifth and sixth somitomeres. Eventually, neural crest originating from the mesencephalon spreads caudally as far as the fourth somitomere, leaving a gap in crest emigration adjacent to the fifth somitomere. The otic placode develops from the surface ectoderm covering the sixth and seventh somitomeres, and the adjacent rhombencephalic neural crest moves around the cranial and caudal edge of the placode. At more caudal levels, rhombencephalic crest cells merge with cervical crest populations to form a continuous sheet over the somites. By the time the anterior neuropore closes, some of the mesencephalic crest cells return from the paraxial mesoderm to spread onto the rostral wall of the optic vesicle and future telencephalon. The segmentation of the mesoderm and patterned distribution of cranial neural crest seen in snapping turtle embryos, further strengthens the argument that the heads of amniotes are derived from a common metameric pattern established early during gastrulation.     

An early marker of axial pattern in the chick embryo and its respecification by retinoic acid.

Chick Ghox 2.9 protein, a homeodomain-containing polypeptide, is first detected in the mid-gastrula stage embryo and its levels increase rapidly in the late gastrula. At this time, the initially narrow band of expression along the primitive streak expands laterally to form a shield-like domain that encompasses almost the entire posterior region of the embryo and extends anteriorly as far as Hensen's node. We have found that this expression domain co-localizes with a morphological feature that consists of a stratum of refractile, thickened mesoderm. Antibody-staining indicates that Ghox 2.9 protein is present in all cells of this mesodermal region. In contrast, expression within the ectoderm overlying the region of refractile mesoderm varies considerably. The highest levels of expression are found in ectoderm near the streak and surrounding Hensen's node, regions that recent fate mapping studies suggest that primarily destined to give rise to neurectoderm. At the definitive streak stage (Hamburger and Hamilton stage 4) the chick embryo is especially sensitive to the induction of axial malformations by retinoic acid. Four hours after the treatment of definitive streak embryos with a pulse of retinoic acid the expression of Ghox 2.9 protein is greatly elevated. This ectopic expression occurs in tissues anterior to Hensen's node, including floor plate, notochord, presumptive neural plate and lateral plate mesoderm, but does not occur in the anteriormost region of the embryo. The ectopic induction of Ghox 2.9 is strongest in ectoderm, and weaker in the underlying mesoderm. Endoderm throughout the embryo is unresponsive. At stage 11, Ghox 2.9 is normally expressed at high levels within rhombomere 4 of the developing hindbrain. In retinoic-acid-treated embryos which have developed to this stage, typical rhombomere boundaries are largely absent. Nevertheless, Ghox 2.9 is still expressed as a discrete band, but one that is widened and displaced to a more anterior position.