Distinct cellular mechanisms of blood vessel fusion in the zebrafish embryo

Although many of the cellular and molecular mechanisms of angiogenesis have been intensely studied [1], little is known about the processes that underlie vascular anastomosis. We have generated transgenic fish lines expressing an EGFP-tagged version of the junctional protein zona occludens 1 (ZO1) to visualize individual cell behaviors that occur during vessel fusion and lumen formation in vivo. These life observations show that endothelial cells (ECs) use two distinct morphogenetic mechanisms, cell membrane invagination and cord hollowing to generate different types of vascular tubes. During initial steps of anastomosis, cell junctions that have formed at the initial site of cell contacts expand into rings, generating a cellular interface of apical membrane compartments, as defined by the localization of the apical marker podocalyxin-2 (Pdxl2). During the cord hollowing process, these apical membrane compartments are brought together via cell rearrangements and extensive junctional remodeling, resulting in lumen coalescence and formation of a multicellular tube. Vessel fusion by membrane invagination occurs adjacent to a preexisting lumen in a proximal to distal direction and is blood-flow dependent. Here, the invaginating inner cell membrane undergoes concomitant apicobasal polarization and the vascular lumen is formed by the extension of a transcellular lumen through the EC, which forms a unicellular or seamless tube.     

Mechanisms of pattern formation in development and evolution

We present a classification of developmental mechanisms that have been shown experimentally to generate pattern and form in metazoan organisms. We propose that all such mechanisms can be organized into three basic categories and that two of these may act as composite mechanisms in two different ways. The simple categories are cell autonomous mechanisms in which cells enter into specific arrangements ('patterns') without interacting, inductive mechanisms in which cell communication leads to changes in pattern by reciprocal or hierarchical alteration of cell phenotypes ('states') and morphogenetic mechanisms in which pattern changes by means of cell interactions that do not change cell states. The latter two types of mechanism can be combined either morphostatically, in which case inductive mechanisms act first, followed by the morphogenetic mechanism, or morphodynamically, in which case both types of mechanisms interact continuously to modify each other's dynamics. We propose that this previously unexplored distinction in the operation of composite developmental mechanisms provides insight into the dynamics of many developmental processes. In particular, morphostatic and morphodynamic mechanisms respond to small changes in their genetic and microenvironmental components in dramatically different ways. We suggest that these differences in 'variational properties' lead to morphostatic and morphodynamic mechanisms being represented to different extents in early and late stages of development and to their contributing in distinct ways to morphological transitions in evolution.     

Concerning one obsolete tradition: Does gastrulation in sponges exist?

The analysis of comparative-embryological and molecular-biological data leads to the conclusion that universal basic mechanisms of morphogenesis occurred first in the evolution of animals in the ancestors of modern sponges and eumetazoans, which served as a basis of different evolution of individual development in Parazoa and Eumetazoa lines. In the former, morphogenesis in early embryogenesis led to formation of the water-current system as a means for capturing and delivery of food particles to different parts of the animal. In the latter, morphogenetic movements manifested themselves as gastrulation, during which the germ layers and the digestive system formed. The morphogenetic movements of cells in Metazoa emerged independently of cell specification. They are primary relative to cell differentiation. The unity of all Metazoa is based on the similarity of mechanisms of morphogenesis rather than on the presence of germ layers.     

Cell-polarity dynamics controls the mechanism of lumen formation in epithelial morphogenesis

Many organs consist of tubes of epithelial cells enclosing a central lumen. How the space of this lumen is generated is a key question in morphogenesis. Two predominant mechanisms of de novo lumen formation have been observed: hollowing and cavitation. In hollowing, the lumen is formed by exocytosis and membrane separation, whereas, in cavitation, the lumen is generated by apoptosis of cells in the middle of the structure [1, 2]. Using MDCK cells in three-dimensional cultures, we found an inverse correlation between polarization efficiency and apoptosis. When cells were grown in collagen, where cells polarized slowly, apoptosis was needed for lumen formation. However, in the presence of Matrigel, which allowed rapid polarization, lumens formed without apoptosis. If polarization in Matrigel was perturbed by blocking formation of the apical surface by RNAi of Cdc42, lumens formed by apoptosis. In a complementary approach, we plated cells at high density so that aggregates formed with little polarity. These aggregates required apoptosis to form lumens, whereas cells plated at low density formed cysts with rapidly polarizing cells and did not need apoptosis to form lumens. The mechanism of lumen formation in the 3D-MDCK model can shift between hollowing and cavitation, depending on cell polarization.     

Mechanisms for removal of developmentally abnormal cells: cell competition and morphogenetic apoptosis

Various cell differentiation signals are tightly linked with apoptotic signals. For example, as a result of somatic mutations, cells within a developing field occasionally receive an altered level of morphogenetic signaling that gives rise to an abnormal cell type. However, these developmentally abnormal cells are frequently removed by activating apoptotic signals. Although such phenomena are crucial for assuring normal development and maintaining a healthy state of various organs, the molecular mechanisms that sense aberrant signals and activate the apoptotic pathway(s) have not fully been investigated. In this review, we discuss recent progress in this area. Cell competition and morphogenetic apoptosis are two kinds of cell death, both of which are mediated by abnormal signaling of Dpp, a member of the TGF-beta superfamily that functions in Drosophila as a morphogen, mitogen and survival factor. Cell competition results in autonomous apoptosis induced by reduced reception of the extracellular survival factor Dpp, while morphogenetic apoptosis is nonautonomous, and is induced by contact of cells receiving different levels of Dpp signaling.     

Internalization of multiple cells during C. elegans gastrulation depends on common cytoskeletal mechanisms but different cell polarity and cell fate regulators

Understanding the links between developmental patterning mechanisms and force-producing cytoskeletal mechanisms is a central goal in studies of morphogenesis. Gastrulation is the first morphogenetic event in the development of many organisms. Gastrulation involves the internalization of surface cells, often driven by the contraction of actomyosin networks that are deployed with spatial precision—both in specific cells and in a polarized manner within each cell. These cytoskeletal mechanisms rely on different cell fate and cell polarity regulators in different organisms. Caenorhabditis elegans gastrulation presents an opportunity to examine the extent to which diverse mechanisms may be used by dozens of cells that are internalized at distinct times within a single organism. We identified 66 cells that are internalized in C. elegans gastrulation, many of which were not known previously to gastrulate. To gain mechanistic insights into how these cells internalize, we genetically manipulated cell fate, cell polarity and cytoskeletal regulators and determined the effects on cell internalization. We found that cells of distinct lineages depend on common actomyosin-based mechanisms to gastrulate, but different cell fate regulators, and, surprisingly, different cell polarity regulators. We conclude that diverse cell fate and cell polarity regulators control common mechanisms of morphogenesis in C. elegans. The results highlight the variety of developmental patterning mechanisms that can be associated with common cytoskeletal mechanisms in the morphogenesis of an animal embryo.     

Vascularization and Development of Cytoarchitectonics in the Human Neocortical Rudiment

The development of cytoarchitectonics of the brain rudiments in mammals is accompanied by the formation of an intracerebral vascular network. The relationship between these two processes is insufficiently clear. We studied the development of blood vessels and cytoarchitectonics in the neocortical rudiment of 6- to 13-week old human embryos. The light and electron microscopy methods were used, as well as histochemical visualization of NADPH-diaphorase in the vessel cells. The endothelium proliferation was evaluated using antibodies to proliferating cell nuclear antigen. Starting from week 8 of development, the tangentially oriented vessels formed a intraneural network in the ventricular zone of the rudiment, which appears to restrict the motility of neuroepithelial cells. The basal membrane was initially absent, and the neuroepithelial cells were in direct contact with the endothelial cells. During week 9 of development, the tangentially oriented vessels appeared in the intermediate zone. Formations similar to glial legs with short regions of the basal membrane adjoined the walls of inter- and intraneural vessels (note that, according to the published data, glial fibrillary acidic protein is not yet visualized at this stage). Angioarchitectonics depended little on the cell population density in different zones of the rudiment; specifically, the cortical plate did not contain tangentially oriented vessels until week 12–13 of development. The data we obtained suggest that the blood vessels fulfill a special morphogenetic function in the developing neocortex.     

Vascularization and development of the cytoarchitecture in the human neocortical rudiment

The development of cytoarchitectonics of the brain rudiments in mammals is accompanied by the formation of an intracerebral vascular network. The relationship between these two processes is insufficiently clear. We studied the development of blood vessels and cytoarchitectonics in the neocortical rudiment of 6- to 13-week old human embryos. The light and electron microscopy methods were used, as well as histochemical visualization of NADPH-diaphorase in the vessel cells. The endothelium proliferation was evaluated using antibodies to proliferating cell nuclear antigen. Starting from week 8 of development, the tangentially oriented vessels formed a intraneural network in the ventricular zone of the rudiment, which appears to restrict the motility of neuroepithelial cells. The basal membrane was initially absent, and the neuroepithelial cells were in direct contact with the endothelial cells. During week 9 of development, the tangentially oriented vessels appeared in the intermediate zone. Formations similar to glial legs with short regions of the basal membrane adjoined the walls of inter- and intraneural vessels (note that, according to the published data, glial fibrillary acidic protein is not yet visualized at this stage). Angioarchitectonics depended little on the cell population density in different zones of the rudiment; specifically, the cortical plate did not contain tangentially oriented vessels until week 12-13 of development. The data we obtained suggest that the blood vessels fulfill a special morphogenetic function in the developing neocortex.     

Evolutionary significance of morphogenetic mechanisms

Different approaches to interpretation of ontogenesis are compared. The importance is emphasized of Ber’s theory of developmental patterns and the law of embryonic similarity. The significance of evolutionary rationalization of morphogenetic mechanisms is discussed (V.N. Beklemishev, V.A. Dogel’ and O.M. Ivanova-Kazas). There are two main pathways of rationalization of morphogenetic mechanisms: by means of epithelial morphogenesis and at the expense of oligocellular primordia (V.N. Beklemishev). When the developmental plans of the taxonomical types are determined, it is important to take into consideration the diversity of morphogenetic mechanisms within a given group in order to reveal morphological spectrums of morphogenetic mechanisms. A comparison of morphogenetic spectrums in different groups provides a wide evolutionary picture and helps us to reconstruct the phylogeny. Influence of morphogenetic mechanisms on the structure of definitive forms is demonstrated: construction technologies determine types of organs, anatomical and histological systems, and even symmetrical features of organisms and their complex parts.     

Epithelial morphogenesis.

The identification of protein factors, such as epimorphin, scatter factor, and activin, that induce epithelial branching and convergent extension-like movements in embryonic tissues are important breakthroughs in our understanding of the role of mesenchyme in epithelial morphogenesis. Moreover, the development of simple in vitro epithelial cell systems that undergo morphogenesis in response to these factors should provide a means to investigate the cellular and molecular bases of the morphogenetic movements themselves. Although many different cellular processes are involved in such morphogenetic behaviors, cell rearrangement is a particularly intriguing one that will be important to study further. Several considerations lead to the prediction that a dynamic regulation of cell-cell adhesion is likely to play a central role in cell rearrangements and epithelial morphogenesis. Ultimately, a greater issue to be addressed is how the different cellular mechanisms participating in epithelial morphogenesis are coordinated and regulated, so as to generate the diverse patterns found in various epithelia.     

The developing neurovascular anatomy of the embryo: a technique of simultaneous evaluation using fluorescent labeling, confocal microscopy, and three-dimensional reconstruction.

The close spatial relationship between peripheral nerves and blood vessels in the adult is well known. However, evidence supporting the congruent development of these structures in embryos remains anecdotal. Neurovascular relationships also have been shown to be conserved in other vertebrates. This homology suggests that either peripheral nerves or blood vessels, or both, might have fundamental morphogenetic roles during embryologic development. Both peripheral nerves and blood vessels have been independently implicated as etiologic agents in the pathogenesis of congenital disabilities, and several congenital anomalies fit their distribution patterns. This article presents a technique for the simultaneous visualization of peripheral nerves and blood vessels at different stages in the developing embryo. The forelimbs of 310 quail embryos were dissected over a 1-year period. Peripheral nerves were labeled with the neural crest and axon antibody, HNK-1, followed by fluorescein-conjugated secondary antibodies. Blood vessels were labeled by a perfusion technique using the fluorescent dye, dioctadecyl-tetramethylindocarbocyanine. Specimens were processed and imaged in whole-mount with confocal microscopy, and images were reconstructed using three-dimensional modeling software. Both nerves and blood vessels seem to undergo a highly stereotypic sequence of development in the embryonic quail forelimb. Furthermore, the existence of a close spatial relationship between nerves and blood vessels suggests either a high degree of developmental interdependence or shared patterning mechanisms. This technique permits further evaluation of the possible role peripheral nerves and blood vessels might play in the pathogenesis of congenital disabilities and provides a starting point for further studies aimed at elucidating the means by which peripheral nerves and blood vessels are patterned in the forelimb of the avian embryo.     

Induction of Invasion and Tubulogenesis in Primary Human Keratinocyte Culture

In this study we used invasion and tubulogenesis assays to measure the morphogenetic potential of primary human keratinocytes in vitro. We also used human embryonic and dermal fibroblasts in coculture with keratinocytes and compared their influence on keratinocyte behavior in these different cell culture systems. The keratinocytes retained morphogenetic potential in vitro. In the presence of embryonic fibroblasts, the keratinocytes formed tubes and invaded collagen gel. A three-dimensional architecture of invasion clusters was reconstructed. We conclude that the cellular mechanisms of invasion and tubulogenesis are similar.     

Bacterial mitotic machineries

Here, we review recent progress that yields fundamental new insight into the molecular mechanisms behind plasmid and chromosome segregation in prokaryotic cells. In particular, we describe how prokaryotic actin homologs form mitotic machineries that segregate DNA before cell division. Thus, the ParM protein of plasmid R1 forms F actin-like filaments that separate and move plasmid DNA from mid-cell to the cell poles. Evidence from three different laboratories indicate that the morphogenetic MreB protein may be involved in segregation of the bacterial chromosome.     

Cell polarity: The missing link in skeletal morphogenesis?

Despite extensive genetic analysis of the dynamic multi-phase process that transforms a small population of lateral plate mesoderm into the mature limb skeleton, the mechanisms by which signaling pathways regulate cellular behaviors to generate morphogenetic forces are not known. Recently, a series of papers have offered the intriguing possibility that regulated cell polarity fine-tunes the morphogenetic process via orienting cell axes, division planes and cell movements. Wnt5a-mediated non-canonical signaling, which may include planar cell polarity, has emerged as a common thread in the otherwise distinct signaling networks that regulate morphogenesis in each phase of limb development. These findings position the limb as a key model to elucidate how global tissue patterning pathways direct local differences in cell behavior that, in turn, generate growth and form.     

Embryonic Tissue Interactions as the Basis for Morphological Change in Evolution

Phenotypic changes over geological time must result from alterationsin the morphogenetic mechanisms associated with organogenesis.Recent advances in our understanding of such mechanisms suggestthat there are certain patterns of metabolic activity and organellesynthesis which are present in many different cell lineagesat different times during embryonic development, and that thisubiquity of genomic potential is available for expression atany time. Microevolutionary sequences of morphological changeprobably result from modulations of quantitative aspects oforganogenesis, e.g., rates of cell proliferation, or cell density.It is also possible that certain macroevolutionary steps ("neomorphs")may result from qualitative changes in the developmental programs,and such events underly "ultimate refinement" of morphologicaladaptation. The selection pressures involved in these differentsequences of morphological change "see" embryonic processesin different ways. This thesis is illustrated with referenceto the evolutionary history of the tetrapod limb.     

Cell polarity

Despite extensive genetic analysis of the dynamic multi-phase process that transforms a small population of lateral plate mesoderm into the mature limb skeleton, the mechanisms by which signaling pathways regulate cellular behaviors to generate morphogenetic forces are not known. Recently, a series of papers have offered the intriguing possibility that regulated cell polarity fine-tunes the morphogenetic process via orienting cell axes, division planes and cell movements. Wnt5a-mediated non-canonical signaling, which may include planar cell polarity, has emerged as a common thread in the otherwise distinct signaling networks that regulate morphogenesis in each phase of limb development. These findings position the limb as a key model to elucidate how global tissue patterning pathways direct local differences in cell behavior that, in turn, generate growth and form.     

Mechanical Stress and Gibberellin: Regulation of Hollowing Induction in the Stem of a Bean Plant, Phaseolus vulgaris L.

In pole bean plants, mechanical stress (MS) inhibited stem elongationand induced radial thickening of the stem. Application of uniconazole,an inhibitor of gibberellin biosynthesis, also reduced stemgrowth but had no effect on stem diameter. Both MS and uniconazolesignificantly reduced hollowing of the first internodes, butonly the former increased ethylene evolution from the firstinternode. Application of GA₃ increased the length of the firstinternode and decreased its diameter in bush bean plants; thiswas accompanied by a significant promotion of stem hollowing.Aminooxyacetic acid (AOA) decreased ethylene evolution fromthe GA₃-treated internodes, though it did not reduce the GA₃-inducedhollowing of the first internodes. Application of GA₃ affectedneither ethylene evolution nor cellulase activity in the firstinternodes of bush bean plants. Application of GA₃ stimulatedmuch greater cell elongation in the center of pith tissue thanin the outer surrounding tissues, suggesting a possible physicalbreakage of the inner cells, which leads the hollowing of beanstems. These results suggest that gibberellin is a factor responsiblefor stem hollowing in bean plants. Because MS is known to reducegibberellin content in bean plants [Suge (1978) Plant Cell Physiol.21: 303] MS may inhibit stem hollowing by reducing the amountof endogenous gibberellin. (Received July 1, 1994; Accepted November 8, 1994)     

How different types of pattern formation mechanisms affect the evolution of form and development

Here we investigate how development and evolution can affect each other by exploring what kind of phenotypic variation is produced by different types of developmental mechanisms. A limited number of developmental mechanisms are capable of pattern formation in development. Two main types have been identified. In morphodynamic mechanisms, induction events and morphogenetic processes, such as simple growth, act at the same time. In morphostatic mechanisms, induction events happen before morphogenetic mechanisms, and thus growth cannot influence the induction of a pattern. We present a study of the variational properties of these developmental mechanisms that can help to understand how and why a developmental mechanism may become involved in the evolution and development of a particular morphological structure. Using existing models of pattern formation in teeth, an extensive simulation analysis of the phenotypic variation produced by different types of developmental mechanisms is performed. The studied properties include the amount and diversity of the phenotypic variation produced, the complexity of the phenotypic variation produced, and the relationship between phenotype and genotype. These variational properties are so different between different types of mechanisms that the relative involvement of these types of mechanisms in evolutionary innovation and in different stages of development can be estimated. In addition, type of mechanism affects the tempo and mode of morphological evolution. These results suggest that the basic principles by which development is organized can influence the likelihood of morphological evolution.     

Distinct signalling pathways regulate sprouting angiogenesis from the dorsal aorta and the axial vein

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is critical to most physiological processes and many pathological conditions. During zebrafish development, angiogenesis expands the axial vessels into a complex vascular network that is necessary for efficient oxygen delivery. Although the dorsal aorta and the axial vein are spatially juxtaposed, the initial angiogenic sprouts from these vessels extend in opposite directions, indicating that distinct cues may regulate angiogenesis of the axial vessels. We found that angiogenic sprouts from the dorsal aorta are dependent on vascular endothelial growth factor A (Vegf-A) signalling, and do not respond to bone morphogenetic protein (Bmp) signals. In contrast, sprouts from the axial vein are regulated by Bmp signalling independently of Vegf-A signals, indicating that Bmp is a vein-specific angiogenic cue during early vascular development. Our results support a paradigm whereby different signals regulate distinct programmes of sprouting angiogenesis from the axial vein and dorsal aorta, and indicate that signalling heterogeneity contributes to the complexity of vascular networks.