Ventral cell rearrangements contribute to anterior-posterior axis lengthening between neurula and tailbud stages in Xenopus laevis

Studies of morphogenesis in early Xenopus embryos have focused primarily on gastrulation and neurulation. Immediately following these stages is another period of intense morphogenetic activity, the neurula-to-tailbud transition. During this period the embryo is transformed from the spherical shape of the early stages into the long, thin shape of the tailbud stages. While gastrulation and neurulation depend largely on active cell rearrangement and cell shape changes in dorsal tissues, we find that the neurula-to-tailbud transition depends in part on activities of ventral cells. Ventral explants of neurula lengthen autonomously as much as the ventral sides of intact embryos, while dorsal explants lengthen less than the dorsal sides of intact embryos. Analyses of cell division, cell shapes, and cell rearrangement by transplantation of labeled cells and by time lapse recordings in live intact embryos concur that cell rearrangements in ventral mesoderm and ectoderm contribute to the autonomous anterior-posterior axis lengthening of ventral explants between neurula and tailbud stages.     

Stereotypical cell division orientation controls neural rod midline formation in zebrafish

The development of multicellular organisms is dependent on the tight coordination between tissue growth and morphogenesis. The stereotypical orientation of cell divisions has been proposed to be a fundamental mechanism by which proliferating and growing tissues take shape. However, the actual contribution of stereotypical division orientation (SDO) to tissue morphogenesis is unclear. In zebrafish, cell divisions with stereotypical orientation have been implicated in both body-axis elongation and neural rod formation, although there is little direct evidence for a critical function of SDO in either of these processes. Here we show that SDO is required for formation of the neural rod midline during neurulation but dispensable for elongation of the body axis during gastrulation. Our data indicate that SDO during both gastrulation and neurulation is dependent on the noncanonical Wnt receptor Frizzled 7 (Fz7) and that interfering with cell division orientation leads to severe defects in neural rod midline formation but not body-axis elongation. These findings suggest a novel function for Fz7-controlled cell division orientation in neural rod midline formation during neurulation.     

A Quantitative Study of Regional and Stage Specific Reaction of Xenopus laevisEmbryonic 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.     

Cloning of cDNAs encoding retinoic acid receptors RAR gamma 1, RAR gamma 2, and a new splicing variant, RAR gamma 3, from Aambystoma mexicanum and characterization of their expression during early development

To analyze retinoic acid (RA) receptor (RAR) expression during early development in the urodele embryo, we have isolated cDNAs for four members of the axolotl (Ambystoma mexicanum) RAR family, namely RAR alpha (NR1B1), aRAR gamma 1 (NR1B3a), aRAR gamma 2 (NR1B3b), and a new splicing variant of aRAR gamma 2, aRAR gamma 3 (NR1B3c), which contains an insertion of five hydrophobic amino acids in the C-terminal region of the DNA binding domain. The temporal expression pattern of the RAR gamma isoforms was established by RT-PCR using total RNA from embryos of different stages. The expression of aRAR gamma 2 coincides with neurulation and is enhanced in the extremities of the embryo's anteroposterior axis. The aRAR gamma 3 is specifically expressed during gastrulation and early neurulation, whereas aRAR gamma 1 is expressed later during organogenesis. Global aRAR gamma 2 mRNA levels, as well as their spatio-temporal expression pattern in the neurula, were not affected by treatment with RA. These results show that several RARs are expressed in the axolotl embryo during early development, and reveal the existence of a new RAR gamma variant.     

The problem of germ layers in sponges (Porifera) and some issues concerning early metazoan evolution

Nowadays the formation of germ layers (endoderm and mesoderm) is associated with gastrulation. The question of whether the cell movements during early embryonic development in sponges (Porifera) are gastrulation as in eumetazoans remains in dispute. Recent data on the histological organization, digestion and embryonic morphogenesis in sponges are analyzed here in an attempt to answer this question. Unique features of these basal Metazoa are the lack of intestinal epithelium, digestive parenchyma or any cell population specialized in digestion. Food particles are captured by cells of almost all types. These data show that sponges have no embryonic layers such as ectoderm or endoderm, characteristic to eumetazoans, and, consequently, no gastrulation. We make an assumption that the formation of germ layers cannot be considered as a recapitulation of events that took place in the common ancestor of Porifera and Eumetazoa. The unity of Metazoa is expressed not in the presence of gastrulation processes per se, but in the universal nature of cell movement mechanisms ensuring various types of morphogenesis, including those underlying gastrulation. It is concluded that metazoan mechanisms of morphogenetic movements must have emerged in the course of evolution prior to the separation of the germ layers like endoderm and ectoderm.     

Shaping and bending of the avian neural plate as analysed with a fluorescent-histochemical marker

Shaping and bending of the neural plate are cardinal events of neurulation. These processes are initiated in avian embryos shortly after the onset of gastrulation and concluded concomitantly with the completion of gastrulation. The epiblast undergoes extensive morphogenetic movements during gastrulation and neurulation, but the directions, distances, rates, mechanisms and roles of such rearrangements are largely unknown. To begin to understand these morphogenetic movements, we have mapped regional displacements of the epiblast by injecting a fluorescent-histochemical marker into selected prenodal, nodal and postnodal levels of the blastoderm. Lateral epiblast regions (600 microns lateral to the midline and consisting primarily of surface epithelium) are displaced craniomedially, medial regions (300 microns lateral to the midline and consisting of neural plate and preingressed mesoderm) predominantly medially, and midline regions (consisting of neural plate and primitive streak) predominantly caudally. Displacements within the avian neural plate parallel those previously described for the amphibian neural plate. Furthermore, similar tissue displacements occur within the prenodal and postnodal levels of the avian epiblast despite the fact that neurulation is occurring in the former and gastrulation in the latter. Finally, our results show that ectodermal rudiments contained within a single cross-sectional level of the embryo are a composite of cells derived from multiple craniocaudal and mediolateral levels. Thus, regional tissue displacements are important events to consider in the analysis of the early morphogenesis of axial and paraxial organ rudiments derived from the epiblast.     

Actomyosin contractility and microtubules drive apical constriction in Xenopus bottle cells

Cell shape changes are critical for morphogenetic events such as gastrulation, neurulation, and organogenesis. However, the cell biology driving cell shape changes is poorly understood, especially in vertebrates. The beginning of Xenopus laevis gastrulation is marked by the apical constriction of bottle cells in the dorsal marginal zone, which bends the tissue and creates a crevice at the blastopore lip. We found that bottle cells contribute significantly to gastrulation, as their shape change can generate the force required for initial blastopore formation. As actin and myosin are often implicated in contraction, we examined their localization and function in bottle cells. F-actin and activated myosin accumulate apically in bottle cells, and actin and myosin inhibitors either prevent or severely perturb bottle cell formation, showing that actomyosin contractility is required for apical constriction. Microtubules were localized in apicobasally directed arrays in bottle cells, emanating from the apical surface. Surprisingly, apical constriction was inhibited in the presence of nocodazole but not taxol, suggesting that intact, but not dynamic, microtubules are required for apical constriction. Our results indicate that actomyosin contractility is required for bottle cell morphogenesis and further suggest a novel and unpredicted role for microtubules during apical constriction.     

Effect of a mechanical tension on the hydration of DNA in fibres.

Fiber X-ray diffraction and measurement of fibre dimensions yield information about the effects of a mechanical tension on hydration of DNA in fibres. At a given relative humidity, the mechanical tension changes the DNA conformation but does not modify the number of water molecules associated to a nucleotide. The number of water molecules per nucleotide necessary to maintain B form decreases for increasing tensions applied to the DNA fibre. Form transitions can be opposed by mechanical tensions; an energy of 1 Kcal per mole of nucleotide pairs is sufficient to prevent the B to A transition.     

Evolution of gastrulation in the ray-finned (actinopterygian) fishes

Sometime before or during the early Mesozoic era, new lineages of actinopterygian (ray-finned) fishes radically transformed their mode of gastrulation. During this evolutionary transformation, yolky endoderm was a hotspot for ontogenetic change. As holoblastic cleavage patterns were modified into meroblastic cleavage patterns, major changes in cell identity specification occurred within the mesendodermal marginal zone, as well as in the superficial epithelium of the embryo. These cellular identity changes resulted in the appearance of two novel extra-embryonic tissues within the embryos of teleostean fishes: the enveloping layer (EVL) and the yolk syncytial layer (YSL). The generation of these extra-embryonic tissues prompted major morphogenetic changes within the Organizer Region. As these evolutionary changes occurred, the outermost cell layer of the Organizer (the Organizer Epithelium) was apparently retained as a signaling center necessary for the establishment of left-right embryonic asymmetry in the embryo. Conserved and derived features of Organizer morphogenesis and gastrulation within ancient lineages of ray-finned fishes provide important insights into how the genetically encoded cell behaviors of early morphogenesis can be altered during the course of evolution. In particular, a highly divergent form of actinopterygian gastrulation, which is found in the annual fishes of South America, demonstrates that no aspect of vertebrate gastrulation is inherently immutable to evolutionary change.     

A role for N-cadherin in mesodermal morphogenesis during gastrulation

Cell adhesion molecules mediate numerous developmental processes necessary for the segregation and organization of tissues. Here we show that the zebrafish biber (bib) mutant encodes a dominant allele at the N-cadherin locus. When knocked down with antisense oligonucleotides, bib mutants phenocopy parachute (pac) null alleles, demonstrating that bib is a gain-of-function mutation. The mutant phenotype disrupts normal cell-cell contacts throughout the mesoderm as well as the ectoderm. During gastrulation stages, cells of the mesodermal germ layer converge slowly; during segmentation stages, the borders between paraxial and axial tissues are irregular and somite borders do not form; later, myotomes are fused. During neurulation, the neural tube is disorganized. Although weaker, all traits present in bib mutants were found in pac mutants. When the distribution of N-cadherin mRNA was analyzed to distinguish mesodermal from neuroectodermal expression, we found that N-cadherin is strongly expressed in the yolk cell and hypoblast in the early gastrula, just preceding the appearance of the bib mesodermal defects. Only later is N-cadherin expressed in the anlage of the CNS, where it is found as a radial gradient in the forming neural plate. Hence, besides a well-established role in neural and somite morphogenesis, N-cadherin is essential for morphogenesis of the mesodermal germ layer during gastrulation.     

Live imaging and morphometric analysis of embryonic development in the ascidian Ciona intestinalis

The ascidian Ciona intestinalis is one of the model organisms of choice for comparative investigations of chordate development and for unraveling the molecular mechanisms underlying morphogenesis and cell fate specification. Taking advantage of the availability of various genetically encoded fluorescent proteins and of defined cis-regulatory elements, we combined transient transgenesis with laser scanning confocal imaging to acquire and quantitate 3D time-lapse data from living Ciona embryos. We used Ciona tissue-specific enhancers to drive expression of spectrally distinct fluorescent protein reporters to label and simultaneously visualize axially and paraxially positioned mesodermal derivatives, as well as neural precursors in individual embryos. We observed morphogenetic movements, without perturbing development, from the early gastrula throughout the larval stage, including gastrulation, neurulation, convergent extension of the presumptive notochord, and tail elongation. These multidimensional data allowed us to establish a reference system of metrics to quantify key developmental events including blastopore closure and muscle extension. The approach we describe can be used to document morphogenetic cell and tissue rearrangements in living embryos and paves the way for a live digitized anatomical atlas of Ciona.     

Symmetry breaking and convergent extension in early chordate development

The initiation of axis, polarity, cell differentiation, and gastrulation in the very early chordate development is due to the breaking of radial symmetry. It is believed that this occurs by an external signal. We suggest instead spontaneous symmetry breaking through the agency of the Turing-Child field. Increased size or decreased diffusivity, both brought about by mitotic activity, cause the spontaneous loss of stability of the homogeneous state and the evolution of the metabolic pattern during development. The polar metabolic pattern is the cause of polar gene expression, polar morphogenesis (gastrulation), and polar mitotic activity. The Turing-Child theory explains not only the spontaneous formation of the invagination in gastrulation but also the coherent cell movement observed in convergence and extension during gastrulation and neurulation. The theory is demonstrated with respect to experimental observations on the early development of fish, amphibian, and the chick. The theory can explain a multitude of experimental details. For example, it explains the splayed polar progression of reduction in the fish blastoderm. Reduction starts on that side of the blastoderm margin, which will initiate invagination several hours later. It progresses toward the blastoderm center and somewhat laterally from this future "dorsal lip". This is precisely as predicted by a Turing-Child system in a circle. And for a fish like zebrafish with a blastoderm that is slightly oval, reduction is observed to progress along the long axis of the ellipse, which is what Turing-Child theory predicts. In general the shape and the chemical nature of the experimental patterns are the same as predicted by the Turing couple (cAMP, ATP). Embryological polarity and convergent extension are based on polar eigenfunction and saddle-shaped eigenfunction, respectively.     

The spatio-temporal distribution of single-stranded breaks in nuclear DNA in sections of clawed toad embryos during gastrulation and neurulation

Spatial and temporal pattern and quantities of nicks in nuclear DNA during gastrulation and neurulation was studied using nick-translation in sections of Xenopus laevis embryos. Specific changes in the number of nicks in different mesoderm and ectoderm regions were detected during embryogenesis. Dorso-ventral gradient of nuclear labelling was observed in mesoderm and inner ectoderm layer of early and middle gastrula. The gradient was inverted during transition from gastrula to neurula. At the same time dorso-ventral (in mesoderm) and ventro-dorsal (in outer ectoderm layer) gradients of nuclear labelling were increased. The intensity of nuclear labelling in all parts of embryo as a whole was remarkably higher during neurulation as compared with gastrulation. Dorso-ventral gradient of nuclear labelling was observed in mesoderm and ectoderm during neurulation. A connection between the nicks and differentiation status of the cells during early embryogenesis in amphibians is suggested.     

A quantitative study of regional and stage specific reaction of African clawed frog embryonic tissues on mechanical stress

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.     

Computational models for mechanics of morphogenesis

In the developing embryo, tissues differentiate, deform, and move in an orchestrated manner to generate various biological shapes driven by the complex interplay between genetic, epigenetic, and environmental factors. Mechanics plays a key role in regulating and controlling morphogenesis, and quantitative models help us understand how various mechanical forces combine to shape the embryo. Models allow for the quantitative, unbiased testing of physical mechanisms, and when used appropriately, can motivate new experimentaldirections. This knowledge benefits biomedical researchers who aim to prevent and treat congenital malformations, as well as engineers working to create replacement tissues in the laboratory. In this review, we first give an overview of fundamental mechanical theories for morphogenesis, and then focus on models for specific processes, including pattern formation, gastrulation, neurulation, organogenesis, and wound healing. The role of mechanical feedback in development is also discussed. Finally, some perspectives aregiven on the emerging challenges in morphomechanics and mechanobiology. Birth Defects Research (Part C) 96:132–152, 2012. © 2012 Wiley Periodicals, Inc.     

Clonal origin of the mammalian forebrain from widespread oriented mixing of early regionalized neuroepithelium precursors

The forebrain is formed by remodeling and growth of the anterior neural plate. This morphogenesis occurs in response to inductive signals during gastrulation and neurulation but is poorly understood at the cellular level. Here, we have used the LaacZ method of single cells labeling to visualize, at E12.5, clones originated at early stages of mouse forebrain development. The largest clones show that single progenitors can give rise to neuroepithelial cells dispersed across the forebrain. A significant fraction of the clones, and even relatively small ones, populated both the diencephalon and the telencephalon, indicating that the clonal separation between diencephalic and telencephalic progenitors is transient and/or partial. However, two groups of large clones, populating either the diencephalon or the telencephalon, dispersed within their respective domains, suggesting an early regionalization between some diencephalic and telencephalic progenitors. Widespread oriented mixing within these territories and then clonal expansion into smaller domains probably follow this initial regionalization. These data are consistent with a model of progressive specification of forebrain domains. We propose that the ordered expansion of early regionalized progenitor pools for the diencephalon and telencephalon could establish a potential link between early inductive signals and forebrain morphogenesis.     

Inhibition of the cell cycle is required for convergent extension of the paraxial mesoderm during Xenopus neurulation

Coordination of morphogenesis and cell proliferation is essential during development. In Xenopus, cell divisions are rapid and synchronous early in development but then slow and become spatially restricted during gastrulation and neurulation. One tissue that transiently stops dividing is the paraxial mesoderm, a dynamically mobile tissue that forms the somites and body musculature of the embryo. We have found that cessation of cell proliferation is required for the proper positioning and segmentation of the paraxial mesoderm as well as the complete elongation of the Xenopus embryo. Instrumental in this cell cycle arrest is Wee2, a Cdk inhibitory kinase that is expressed in the paraxial mesoderm from mid-gastrula stages onwards. Morpholino-mediated depletion of Wee2 increases the mitotic index of the paraxial mesoderm and this results in the failure of convergent extension and somitogenesis in this tissue. Similar defects are observed if the cell cycle is inappropriately advanced by other mechanisms. Thus, the low mitotic index of the paraxial mesoderm plays an essential function in the integrated cell movements and patterning of this tissue.     

Inductive interactions in the spatial and temporal restriction of lens-forming potential in embryonic ectoderm of Xenopus laevis

The process of lens cell determination in amphibians is currently viewed as one involving a series of inductive interactions. On the basis of previous investigations, these interactions are thought to begin during gastrulation when the presumptive foregut endoderm and then the heart mesoderm come into contact with the presumptive lens ectoderm. This earlier period of induction is followed by the later interaction of the optic vesicle with the lens-forming ectoderm. Transplantation experiments were performed to determine the relative significance of the early and later periods of induction in the process of lens cell determination in the anuran Xenopus laevis. Various ectodermal tissues were transplanted either into the lens-forming region of open neural plate stage host embryos or over the newly formed optic vesicle of later neurula stage embryos. All transplanted tissues were labeled with the intracellular marker horseradish peroxidase to assess the exact origins of any induced lens structures. The results indicate that all nonneural ectodermal tissues have some lens-forming potential early during gastrulation; however, this potential is restricted to the lens-forming region, and perhaps nearby regions, later in development during the time of neurulation. Furthermore, the results show that the optic vesicle is not a substantial inductor of the lens in tissues that have not been previously exposed to the earlier series of inductive interactions that take place during gastrulation and neurulation. Since the optic vesicle does not appear to be a sufficient inductor of the lens, these earlier inductive interactions are, therefore, essential in the process of lens cell determination in Xenopus. These earlier inductive interactions lead to a steady increase in what may be called a lens-forming bias in the presumptive lens ectoderm during this period of development. The eventual loss in the ability of nonlens ventral ectoderm to respond to these lens inductors is presumably the result of other determinative processes that occur in this tissue.     

A Relevant Method for Estimating Cell Cycle of Developing Cell: The Conspicuous Lengthening of Ts during the Early Neural Development of Cynops Embryo

A new method for estimating cell cycle was proposed and the cell cycles of the presumptive neural cells of Cynops embryo from the gastrula to neurula stages were estimated. Up to the onset of gastrulation, Tg₂ and Tg₁ became recognizable and Ts lengthened more than 10 times of that in the morula stage. The respective phases of cell cycle, espetially Ts became prominently longer as gastrulation and neurulation proceeded. However, the Ts retained a correlation with Tgc as expressed in the following regression equation, Ts=0.795Tgc–0.090, through the early development of presumptive neural cells.