Potentiation by the lithium ion of morphogenetic responses to a Xenopus inducing factor

We have cultured explants of Xenopus blastular animal cap tissue from embryos that had received an earlier treatment with LiCl and from their untreated siblings, in various concentrations of XTC-cell-derived mesoderm-inducing factor (XTC-MIF, Smith, 1987; Smith et al. 1988). The pretreatment with lithium that we used transforms later morphogenesis in the whole embryo to give radialized body forms with anterior/dorsal levels of structure grossly over-represented. In addition, animal caps from 'Li+' embryos were allowed to develop without exposure to in vitro MIF (Li+ controls) and compared with normal uninduced control explants, and explants were made from normal early blastulae but given various initial treatments with LiCl in culture. The results confirm that the lithium ion itself will not induce mesoderm in competent, animal cap tissue of Xenopus. It does, however, enhance the responsiveness of this tissue to XTC-MIF, in a way that parallels its recently reported effect in the case of another mesoderm inducer of different character, bFGF (Slack et al. 1988). The effects observed are sufficient to imply that the altered body pattern that follows lithium treatment, in whole embryos, could be caused by modulation of the responses to an unaltered pattern of in situ inductive stimuli. We also observe evidence that appreciable inductive signals reach animal pole tissue beyond the limits of mesoderm formation in normal development. Relatively low concentrations of MIF prevent the development of an epidermis-specific marker in dissociated blastular animal cap cells (Symes et al. 1988). When such experiments are repeated in relation to the lithium pretreatment of embryos, such treatment is seen to have sensitized the cell population, so that the MIF concentration range that assures complete suppression of the marker is reduced. The results are discussed in relation to induction considered as pattern formation.     

Genetic dissection of nodal function in patterning the mouse embryo

Loss-of-function analysis has shown that the transforming growth factor-like signaling molecule nodal is essential for mouse mesoderm development. However, definitive proof of nodal function in other developmental processes in the mouse embryo has been lacking because the null mutation blocks gastrulation. We describe the generation and analysis of a hypomorphic nodal allele. Mouse embryos heterozygous for the hypomorphic allele and a null allele undergo gastrulation but then display abnormalities that fall into three distinct mutant phenotypic classes, which may result from expression levels falling below critical thresholds in one or more domains of nodal expression. Our analysis of each of these classes provides conclusive evidence for nodal-mediated regulation of several developmental processes in the mouse embryo, beyond its role in mesoderm formation. We find that nodal signaling is required for correct positioning of the anteroposterior axis, normal anterior and midline patterning, and the left-right asymmetric development of the heart, vasculature, lungs and stomach.     

Low proliferative and high migratory activity in the area of Brachyury expressing mesoderm progenitor cells in the gastrulating rabbit embryo

General mechanisms initiating the gastrulation process in early animal development are still elusive, not least because embryonic morphology differs widely among species. The rabbit embryo is revived here as a model to study vertebrate gastrulation, because its relatively simple morphology at the appropriate stages makes interspecific differences and similarities particularly obvious between mammals and birds. Three approaches that centre on mesoderm specification as a key event at the start of gastrulation were chosen. (1) A cDNA fragment encoding 212 amino acids of the rabbit Brachyury gene was cloned by RT-PCR and used as a molecular marker for mesoderm progenitors. Whole-mount in situ hybridisation revealed single Brachyury-expressing cells in the epiblast at 6.2 days post conception, i.e. several hours before the first ingressing mesoderm cells can be detected histologically. With the anterior marginal crescent as a landmark, these mesoderm progenitors are shown to lie in a posterior quadrant of the embryonic disc, which we call the posterior gastrula extension (PGE), for reasons established during the following functional analysis. (2) Vital dye (DiI) labelling in vitro suggests that epiblast cells arrive in the PGE from anterior parts of the embryonic disc and then move within this area in a complex pattern of posterior, centripetal and anterior directions to form the primitive streak. (3) BrdU labelling shows that proliferation is reduced in the PGE, while the remaining anterior part of the embryonic disc contains several areas of increased proliferation. These results reveal similarities with the chick with respect to Brachyury expression and cellular migration. They differ, however, in that local differences in proliferation are not seen in the pre-streak avian embryo. Rather, rabbit epiblast cells start mesoderm differentiation in a way similar to Drosophila, where a transient downregulation of proliferation initiates mesoderm differentiation and, hence, gastrulation.     

Xenopus mesoderm induction: evidence for early size control and partial autonomy for pattern development by onset of gastrulation

Experiments are described that examine the state of organisation of the presumptive mesoderm and ectoderm of the Xenopus embryo at stages up to the onset of gastrulation. It is shown that a process during blastula stages, establishing the normal proportions in which this cell population is partitioned to found the two outer 'germ layers', has a positive regulative property. An operation has been performed to excise the yolky endodermal core, at the beginning of gastrulation, leaving only the presumptive territories of mesoderm, neural tissue, epidermis and supra-blastoporal endoderm. This reveals that by this time a stable capacity exists within the induced tissue to express the craniocaudal sequence of the normal pattern, including the proper numbers of somite segments. The mediolateral organisation of such body patterns is however abnormal. The relevance of the observations to understanding mechanisms of axial pattern control is discussed.     

Expression of the T-box gene Eomesodermin during early mouse development.

We report the expression pattern of a murine homolog of the Xenopus laevis T-box gene Eomesodermin. mEomes expression is first detected in the extra-embryonic ectoderm prior to gastrulation, and persists there until head-fold stages. In the embryo proper, mEomes is expressed throughout the early primitive streak, nascent mesoderm and in the anterior visceral endoderm. Although mEomes expression disappears from the embryo at late-streak stages, a second domain of mEomes expression is observed in the telencephalon beginning around E10.5.     

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.     

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.     

Control of early anterior-posterior patterning in the mouse embryo by TGF-beta signalling

Prior to gastrulation the mouse embryo exists as a symmetrical cylinder consisting of three tissue layers. Positioning of the future anterior-posterior axis of the embryo occurs through coordinated cell movements that rotate a pre-existing proximal-distal (P-D) axis. Overt axis formation becomes evident when a discrete population of proximal epiblast cells become induced to form mesoderm, initiating primitive streak formation and marking the posterior side of the embryo. Over the next 12-24 h the primitive streak gradually elongates along the posterior side of the epiblast to reach the distal tip. The most anterior streak cells comprise the 'organizer' region and include the precursors of the so-called 'axial mesendoderm', namely the anterior definitive endoderm and prechordal plate mesoderm, as well as those cells that give rise to the morphologically patent node. Signalling pathways controlled by the transforming growth factor-beta ligand nodal are involved in orchestrating the process of axis formation. Embryos lacking nodal activity arrest development before gastrulation, reflecting an essential role for nodal in establishing P-D polarity by generating and maintaining the molecular pattern within the epiblast, extraembryonic ectoderm and the visceral endoderm. Using a genetic strategy to manipulate temporal and spatial domains of nodal expression reveals that the nodal pathway is also instrumental in controlling both the morphogenetic movements required for orientation of the final axis and for specification of the axial mesendoderm progenitors.     

Sequential allocation and global pattern of movement of the definitive endoderm in the mouse embryo during gastrulation

During mouse gastrulation, endoderm cells of the dorsal foregut are recruited ahead of the ventral foregut and move to the anterior region of the embryo via different routes. Precursors of the anterior-most part of the foregut and those of the mid- and hind-gut are allocated to the endoderm of the mid-streak-stage embryo, whereas the precursors of the rest of the foregut are recruited at later stages of gastrulation. Loss of Mixl1 function results in reduced recruitment of the definitive endoderm, and causes cells in the endoderm to remain stationary during gastrulation. The observation that the endoderm cells are inherently unable to move despite the expansion of the mesoderm in the Mixl1-null mutant suggests that the movement of the endoderm and the mesoderm is driven independently of one another.     

Early specification for body position in mes-endodermal regions of an amphibian embryo

Specification for development of the body pattern in the amphibian embryo has usually been thought of as a prolonged process, initiated from an ooplasmic localisation of some kind in what will become the dorsal-anterior midline. The evidence has been interpreted as suggesting that this initial localisation is centred in what will become anterior endoderm, but gives rise by an inductive process in early blastula stages to an overlying organising centre which eventually controls the genesis of mesodermal pattern. Neurectodermal development (especially, the position and pattern of the central nervous system) is seen as controlled considerably later, by inductive signals from submigrating mesoderm at gastrulation. Current work tends to confirm that this sequence of inductive influences can occur at least in experimental situations. It also suggests, however, that in the normal development of the rather small egg of Xenopus, genesis of positional cues that specify the body pattern contributions within the more vegetal material (mes-endoderm) is a rather rapid, widespread and direct consequence of events occurring in the interval between fertilisation and cleavage. Possible molecular bases of early nuclear responses to position within egg material, and the more problematic nature of the positional system itself, are discussed.     

Expression of XMyoD protein in early Xenopus laevis embryos.

A monoclonal antibody specific for Xenopus MyoD (XMyoD) has been characterized and used to describe the pattern of expression of this myogenic factor in early frog development. The antibody recognizes an epitope close to the N terminus of the products of both XMyoD genes, but does not bind XMyf5 or XMRF4, the other two myogenic factors that have been described in Xenopus. It reacts in embryo extracts only with XMyoD, which is extensively phosphorylated in the embryo. The distribution of XMyoD protein, seen in sections and whole-mounts, and by immunoblotting, closely follows that of XMyoD mRNA. XMyoD protein accumulates in nuclei of the future somitic mesoderm from the middle of gastrulation. In neurulae and tailbud embryos it is expressed specifically in the myotomal cells of the somites. XMyoD is in the nucleus of apparently every cell in the myotomes. It accumulates first in the anterior somitic mesoderm, and its concentration then declines in anterior somites from the tailbud stage onwards.     

The anterior-posterior axis emerges respecting the morphology of the mouse embryo that changes and aligns with the uterus before gastrulation

BACKGROUND: When the anterior-posterior axis of the mouse embryo becomes explicit at gastrulation, it is almost perpendicular to the long uterine axis. This led to the belief that the uterus could play a key role in positioning this future body axis. RESULTS: Here, we demonstrate that when the anterior-posterior axis first emerges it does not respect the axes of the uterus but, rather, the morphology of the embryo. Unexpectedly, the emerging anterior-posterior axis is initially aligned not with the long, but the short axis of the embryo. Then whether the embryo develops in vitro or in utero, the anterior-posterior axis becomes aligned with the long axis of embryo just prior to gastrulation. Of three mechanisms that could account for this apparent shift in anterior-posterior axis orientation-cell migration, spatial change of gene expression, or change in embryo shape-lineage tracing studies favor a shape change accompanied by restriction of the expression domain of anterior markers. This property of the embryo must be modulated by interactions with the uterus as ultimately the anterior-posterior and long axes of the embryo align with the left-right uterine axis. CONCLUSIONS: The emerging anterior-posterior axis relates to embryo morphology rather than that of the uterus. The apparent shift in its orientation to align with the long embryonic axis and with the uterus is associated with a change in embryo shape and a refinement of anterior gene expression pattern. This suggests an interdependence between anterior-posterior gene expression, the shape of the embryo, and the uterus.     

Rostral paraxial mesoderm regulates refinement of the eye field through the bone morphogenetic protein (BMP) pathway

The eye field is initially a large single domain at the anterior end of the neural plate and is the first indication of optic potential in the vertebrate embryo. During the course of development, this domain is subject to interactions that shape and refine the organogenic field. The action of the prechordal mesoderm in bisecting this single region into two bilateral domains has been well described, however the role of signalling interactions in the further restriction and refinement of this domain has not been previously characterised. Here we describe a role for the rostral cephalic paraxial mesoderm in limiting the extent of the eye field. The anterior transposition of this mesoderm or its ablation disrupted normal development of the eye. Importantly, perturbation of optic vesicle development occurred in the absence of any detectable changes in the pattern of neighbouring regions of the neural tube. Furthermore, negative regulation of eye development is a property unique to the rostral paraxial mesoderm. The rostral paraxial mesoderm expresses members of the bone morphogenetic protein (BMP) family of signalling molecules and manipulation of endogenous BMP signalling resulted in abnormalities of the early optic primordia.