Early tissue interactions leading to embryonic lens formation in Xenopus laevis

Our previous research has demonstrated that lens induction in Xenopus laevis requires inductive interactions prior to contact with the optic vesicle, which classically had been thought to be the major lens inductor. The importance of these early interactions has been verified by demonstrating that lens ectoderm is specified by the time it comes into contact with the optic vesicle. It has been argued that the tissues which underlie the presumptive lens ectoderm during gastrulation and neurulation, dorsolateral endoderm and mesoderm, are the primary early inductors. We show here, however, that these tissues alone cannot elicit lens formation in Xenopus ectoderm. Evidence is presented that presumptive anterior neural plate tissue (which includes the early eye rudiment) is an essential early lens inductor in Xenopus. The presence of dorsolateral mesoderm appears to enhance this response. These findings support a model in which an essential inductive signal passes through the plane of ectoderm during gastrula and early neurula stages from presumptive anterior neural tissue to the presumptive lens ectoderm. Since there is evidence for such interactions within a tissue layer in mesodermal and neural induction as well, this may be a general feature of the initial stages of determination of many tissues.     

Gastrulation and larval pattern in Xenopus after blastocoelic injection of a Xenopus-derived inducing factor: experiments testing models for the normal organization of mesoderm

When a Xenopus XTC cell-derived mesoderm-inducing factor (MIF) is injected into the blastocoel of Xenopus embryos before gastrulation, they develop almost normally until just after the onset of mesoderm involution at the internal blastoporal lip. Cells from the entire lining of the blastocoel roof and inner marginal zone then undergo a synchronous, sudden change of contact and arrangement which resembles the transformation undergone by normal mesoderm at its time of involution at the vegetal edge of the marginal zone. We describe a dose-dependent spectrum of subsequent abnormalities in gastrulation and, in cases where gastrulation partially recovers, in the resulting larval pattern. Because of such recovery, embryos injected with widely different doses may appear equally abnormal at the early gastrula stage but very different by control larval stages. Extra spinocaudal axial patterns, in the area of ectopic mesoderm, are seen after MIF doses that just permit recovery of gastrulation. The sudden cellular transformation corresponding to involution, in the ectopically specified mesoderm, spreads throughout the animal cap within 15 min in individuals, at a time significantly later than the earliest normal transformation in the marginal zone. No systematic alteration could, however, be detected in its timing, in relation to a 250-fold range of injected MIF concentration or a 3.5-hr difference in time of injection. The severity of the effects on final embryonic pattern is largely independent of the blastular stage of injections. Splitting of the total injected dose into two, separated by 2 to 3 hr of blastular development, reveals that the degree of effect on gastrulation and patterning depends only upon the highest experienced concentration at any time before response. When fibroblast growth factor (bFGF), a different effective mesoderm inducer, is similarly injected, a similar abnormal cell behavior and ectopic mesoderm formation are seen, but beginning only at midgastrular stages some 1.5 hr beyond that characteristic of XTC-MIF. The findings are introduced and discussed in terms of models for the natural organization of the time course of gastrulation and mesodermal pattern.     

The restrictive effect of early exposure to lithium upon body pattern in Xenopus development, studied by quantitative anatomy and immunofluorescence

We have carried out an anatomical study of Xenopus larval and gastrula stages resulting from treatment of synchronous early blastulae for brief periods with Li+. We confirm the proposal that such treatment causes a particular transformation, and partial elimination, of the normal body pattern. Coordinated restriction of pattern, without appreciable loss of cell number, is seen in all three germ layers. The distortion has been investigated by quantitative study of mesoderms at a standard stage, in relation to the normal fate map for mesoderm, and with the help of immunofluorescence on sections for somitic muscle and for blood. In the extreme syndrome, mesoderm arises from all around the blastula as usual, but is symmetrical and corresponds to that arising near the dorsal/anterior meridian of the normally specified egg or embryo with a large posterior subset of the normal pattern values thus missing. The effect is independent of any inhibition of archenteron formation or mesoderm migration (i.e. the cell mechanics of gastrulation) incurred by the treatment. It is also quite separate from a syndrome caused by more prolonged exposure to Li+ during gastrulation. A small, but distinctive, anterior pattern region is also not expressed and, anomalously in relation to their general nature, these forms differentiate considerable blood tissue. We consider the implications of some details of the pattern restriction for our understanding of interaction in the normal development and propose that the Li+ embryo is likely to be useful as a specific 'differential screen', in relation to the normal, during the search for those gene products that mediate initial regionalization of the body.     

Specification of pharyngeal endoderm is dependent on early signals from axial mesoderm.

The development of taste buds is an autonomous property of the pharyngeal endoderm, and this inherent capacity is acquired by the time gastrulation is complete. These results are surprising, given the general view that taste bud development is nerve dependent, and occurs at the end of embryogenesis. The pharyngeal endoderm sits at the dorsal lip of the blastopore at the onset of gastrulation, and because this taste bud-bearing endoderm is specified to make taste buds by the end of gastrulation, signals that this tissue encounters during gastrulation might be responsible for its specification. To test this idea, tissue contacts during gastrulation were manipulated systematically in axolotl embryos, and the subsequent ability of the pharyngeal endoderm to generate taste buds was assessed. Disruption of both putative planar and vertical signals from neurectoderm failed to prevent the differentiation of taste buds in endoderm. However, manipulations of contact between presumptive pharyngeal endoderm and axial mesoderm during gastrulation indicate that signals from axial mesoderm (the notochord and prechordal mesoderm) specify the pharyngeal endoderm, conferring upon the endoderm the ability to autonomously differentiate taste buds. These findings further emphasize that despite the late differentiation of taste buds, the tissue-intrinsic mechanisms that generate these chemoreceptive organs are set in motion very early in embryonic development.     

Self-organization in the determination of the size of the axial structures in the embryogenesis of the clawed toad

Experiments were performed using X. laevis embryos during gastrulation and neurulation (stages 10, 11 1/2, 12 1/2, 13 1/2, 15 and 18). Part of presumptive epidermis and lateral plate mesoderm was removed, and embryos raised until stage 25. The size of axial structures (notochord, somite mesoderm, central nervous system) was determined using serial histological sections and compared with that of control embryos. In experimental embryos, the size of axial structures was decreased. Until a specific stage of development, close correlation was found between the volume of embryonic compartment corresponding to a particular, structure and the volume of presumptive epidermis and lateral plate mesoderm. This stage is individual for each axial organ: middle gastrula (stage 11 1/2) for notochord, late gastrula (stage 12 1/2) for somite mesoderm, and late neurula (stage 18) for central nervous system. This data suggest that differentiation pattern of ecto-mesodermal rudiment is subject to regulation during gastrulation-neurulation, and subdivision of ectoderm and mesoderm into axial and non-axial tissues is a self-organizing process.     

Planar and vertical induction of anteroposterior pattern during the development of the amphibian central nervous system

In amphibians and other vertebrates, neural development is induced in the ectoderm by signals coming from the dorsal mesoderm during gastrulation. Classical embryological results indicated that these signals follow a “vertical” path, from the involuted dorsal mesoderm to the overlying ectoderm. Recent work with the frog Xenopus laevis, however, has revealed the existence of “planar” neural-inducing signals, which pass within the continuous sheet or plane of tissue formed by the dorsal mesoderm and presumptive neurectoderm. Much of this work has made use of Keller explants, in which dorsal mesoderm and ectoderm are cultured in a planar configuration with contact along only a single edge, and vertical contact is prevented. Planar signals can induce the full anteroposterior (A-P) extent of neural pattern, as evidenced in Keller explants by the expression of genes that mark specific positions along the A-P axis. In this review, classical and modern molecular work on vertical and planar inductionwill be discussed. This will be followed by a discussion of various models for vertical induction and planar induction. It has been proposed that the A-P pattern in the nervous system is derived from a parallel pattern of inducers in the dorsal mesoderm which is “imprinted” vertically onto the overlying ectoderm. Since it is now known that planar signals can also induce A-P neural pattern, this kind of model must be reassessed. The study of planar induction of A-P pattern in Xenopus embryos provides a simple, manipulable, two-dimensional system in which to investigate pattern formation. © 1993 John Wiley & Sons, Inc.     

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.     

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.     

The specification of heart mesoderm occurs during gastrulation in Xenopus laevis

The establishment of heart mesoderm during Xenopus development has been examined using an assay for heart differentiation in explants and explant combinations in culture. Previous studies using urodele embryos have shown that the heart mesoderm is induced by the prospective pharyngeal endoderm during neurula and postneurula stages. In this study, we find that the specification of heart mesoderm must begin well before the end of gastrulation in Xenopus embryos. Explants of prospective heart mesoderm isolated from mid- or late neurula stages were capable of heart formation in nearly 100% of cases, indicating that the specification of heart mesoderm is complete by midneurula stages. Moreover, inclusion of pharyngeal endoderm had no statistically significant effect upon either the frequency of heart formation or the timing of the initiation of heartbeat in explants of prospective heart mesoderm isolated after the end of gastrulation. When the superficial pharyngeal endoderm was removed at the beginning of gastrulation, experimental embryos formed hearts, as did explants of prospective heart mesoderm from such embryos. These results indicate that the inductive interactions responsible for the establishment of heart mesoderm occur prior to the end of gastrulation and do not require the participation of the superficial pharyngeal endoderm.     

Mesoderm differentiation in explants of carp embryos

An analysis of carp blastoderm development was carried out in culture after isolation from the yolk cell and its yolk syncytial layer (YSL). The blastoderms were separated from the YSL at four different stages of embryogenesis: the blastula, early epiboly, early gastrula and late gastrula stages. Absence of the YSL in explants was checked by scanning electron microscopy. From observations of living embryos and histological examination of tissues which were formed in explants from all stages studied it was observed that they contained notochordal, muscle and neural tissue as signs of dorsal types of differentiation. Only in explants from the early and late gastrula stages were histotypical tissues organized in an embryonic-like body pattern. The data indicate that mesoderm differentiation in fish embryos is independent from the YSL, contrary to normal pattern formation which needs the presence of the YSL before the onset of gastrulation.     

Xenopus fibrillin regulates directed convergence and extension

Fibrillin-based human diseases such as Marfan syndrome and congenital contractural arachnodactyly implicate fibrillins in the function and homeostasis of multiple adult tissues. Fibrillins are also expressed in embryos, but no early developmental role has been described for these proteins. We use three independent methods to reveal a role for Xenopus fibrillin (XF) at gastrulation. First, expressing truncated forms of XF in the embryo leads to failure of gastrulation concomitant with a dominant-negative effect on native fibrillin fibril assembly. Expressing truncated XF also inhibits normal progression of the patterned, polarized cell motility that drives convergence and extension at gastrulation and perturbs directed extension in cultured explants of dorsal mesoderm. Second, injection of a synthetic peptide encoding a cell-binding domain of XF into midgastrula embryos causes acute failure of gastrulation associated with defective fibrillin fibril assembly. These injections also reveal a critical role for this peptide in the fibril assembly process. Third, morpholino-mediated knockdown of translation of XF in the embryo also perturbs normal gastrulation and directed extension. Together, these data show that native Xenopus fibrillin is essential for the process of directed convergent extension in presumptive notochord at gastrulation.     

Clonal and molecular analysis of the prospective anterior neural boundary in the mouse embryo

In the mouse embryo the anterior ectoderm undergoes extensive growth and morphogenesis to form the forebrain and cephalic non-neural ectoderm. We traced descendants of single ectoderm cells to study cell fate choice and cell behaviour at late gastrulation. In addition, we provide a comprehensive spatiotemporal atlas of anterior gene expression at stages crucial for anterior ectoderm regionalisation and neural plate formation. Our results show that, at late gastrulation stage, expression patterns of anterior ectoderm genes overlap significantly and correlate with areas of distinct prospective fates but do not define lineages. The fate map delineates a rostral limit to forebrain contribution. However, no early subdivision of the presumptive forebrain territory can be detected. Lineage analysis at single-cell resolution revealed that precursors of the anterior neural ridge (ANR), a signalling centre involved in forebrain development and patterning, are clonally related to neural ectoderm. The prospective ANR and the forebrain neuroectoderm arise from cells scattered within the same broad area of anterior ectoderm. This study establishes that although the segregation between non-neural and neural precursors in the anterior midline ectoderm is not complete at late gastrulation stage, this tissue already harbours elements of regionalisation that prefigure the later organisation of the head.