Development of polyembryonic insects: a major departure from typical insect embryogenesis

 The parasitic wasp Copidosoma floridanum represents the most extreme form of polyembryonic development known, forming up to 2000 embryos from a single egg. To understand the mechanisms of embryonic patterning in polyembryonic wasps and the evolutionary changes that led to this form of development we have analyzed embryonic development at the cellular level using confocal and scanning electron microscopy. C. floridanum embryogenesis can be divided into three phases: (1) early cleavage that leads to formation of a primary morula, (2) a proliferative phase that involves partitioning of embryonic cells into thousands of morulae, and (3) morphogenesis whereby individual embryos develop into larvae. This developmental program represents a major departure from typical insect embryogenesis, and we describe several features of morphogenesis unusual for insects. The early development of polyembryonic wasps, which likely evolved in association with a shift in life history to endoparasitism, shows several analogies with mammalian embryogenesis, including early separation of extraembryonic and embryonic cell lineages, formation of a morula and embryonic compaction. However, the late morphogenesis of polyembryonic wasps proceeds in a fashion conserved in all insects. Collectively, this suggests a lack of developmental constraints in early development, but a strong conservation of the phylotypic stage. Received: 27 June 1997 / Accepted: 11 January 1998     

Animal egg as evolutionary innovation: a solution to the "embryonic hourglass" puzzle

The evolutionary origin of the egg stage of animal development presents several difficulties for conventional developmental and evolutionary narratives. If the egg's internal organization represents a template for key features of the developed organism, why can taxa within a given phylum exhibit very different egg types, pass through a common intermediate morphology (the so-called "phylotypic stage"), only to diverge again, thus exemplifying the embryonic "hourglass"? Moreover, if different egg types typically represent adaptations to different environmental conditions, why do birds and mammals, for example, have such vastly different eggs with respect to size, shape, and postfertilization dynamics, whereas all these features are more similar for ascidians and mammals? Here, I consider the possibility that different body plans had their origin in self-organizing physical processes in ancient clusters of cells, and suggest that eggs represented a set of independent evolutionary innovations subsequently inserted into the developmental trajectories of such aggregates. I first describe how "dynamical patterning modules" (DPMs) associations between components of the metazoan developmental-genetic toolkit and certain physical processes and effects may have organized primitive animal body plans independently of an egg stage. Next, I describe how adaptive specialization of cells released from such aggregates could have become "proto-eggs," which regenerated the parental cell clusters by cleavage, conserving the characteristic DPMs available to a lineage. Then, I show how known processes of cytoplasmic reorganization following fertilization are often based on spontaneous, self-organizing physical effects ("egg-patterning processes": EPPs). I suggest that rather than acting as developmental blueprints or prepatterns, the EPPs refine the phylotypic body plans determined by the DPMs by setting the boundary and initial conditions under which these multicellular patterning mechanisms operate. Finally, I describe how this new perspective provides a resolution to the embryonic hourglass puzzle.     

Identification of novel genes affecting mesoderm formation and morphogenesis through an enhanced large scale functional screen in Xenopus

The formation of mesoderm is an important developmental process of vertebrate embryos, which can be broken down into several steps; mesoderm induction, patterning, morphogenesis and differentiation. Although mesoderm formation in Xenopus has been intensively studied, much remains to be learned about the molecular events responsible for each of these steps. Furthermore, the interplay between mesoderm induction, patterning and morphogenesis remains obscure. Here, we describe an enhanced functional screen in Xenopus designed for large-scale identification of genes controlling mesoderm formation. In order to improve the efficiency of the screen, we used a Xenopus tropicalis unique set of cDNAs, highly enriched in full-length clones. The screening strategy incorporates two mesodermal markers, Xbra and Xmyf-5, to assay for cell fate specification and patterning, respectively. In addition we looked for phenotypes that would suggest effects in morphogenesis, such as gastrulation defects and shortened anterior-posterior axis. Out of 1728 full-length clones we isolated 82 for their ability to alter the phenotype of tadpoles and/or the expression of Xbra and Xmyf-5. Many of the clones gave rise to similar misexpression phenotypes (synphenotypes) and many of the genes within each synphenotype group appeared to be involved in similar pathways. We determined the expression pattern of the 82 genes and found that most of the genes were regionalized and expressed in mesoderm. We expect that many of the genes identified in this screen will be important in mesoderm formation.     

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.     

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.     

Wnt signal transduction pathways

The Wnt signaling pathway is an ancient and evolutionarily conserved pathway that regulates crucial aspects of cell fate determination, cell migration, cell polarity, neural patterning and organogenesis during embryonic development. The Wnts are secreted glycoproteins and comprise a large family of nineteen proteins in humans hinting to a daunting complexity of signaling regulation, function and biological output. To date major signaling branches downstream of the Fz receptor have been identified including a canonical or Wnt/β-catenin dependent pathway and the non-canonical or β-catenin-independent pathway which can be further divided into the Planar Cell Polarity and the Wnt/Ca²⁺ pathways, and these branches are being actively dissected at the molecular and biochemical levels. In this review, we will summarize the most recent advances in our understanding of these Wnt signaling pathways and the role of these pathways in regulating key events during embryonic patterning and morphogenesis.     

Molecular signaling in zebrafish development and the vertebrate phylotypic period

SUMMARY During development vertebrate embryos pass through a stage where their morphology is most conserved between species, the phylotypic period (approximately the pharyngula). To explain the resistance to evolutionary changes of this period, one hypothesis suggests that it is characterized by a high level of interactions. Based on this hypothesis, we examined protein–protein interactions, signal transduction cascades and miRNAs over the course of zebrafish development, and the conservation of expression of these genes in mouse development. We also investigated the characteristics of genes highly expressed before or during the presumed phylotypic period. We show that while there is a high diversity of interactions during the phylotypic period (protein–DNA, RNA–RNA, cell–cell, and between tissues), which is well conserved with mouse, there is no clear difference with later, more morphologically divergent, stages. We propose that the phylotypic period may rather be the expression at the morphological level of strong conservation of molecular processes earlier in development.     

WNTs in the vertebrate nervous system: from patterning to neuronal connectivity

WNT signalling has a key role in early embryonic patterning through the regulation of cell fate decisions, tissue polarity and cell movements. In the nervous system, WNT signalling also regulates neuronal connectivity by controlling axon pathfinding, axon remodelling, dendrite morphogenesis and synapse formation. Studies, from invertebrates to mammals, have led to a considerable understanding of WNT signal transduction pathways. This knowledge provides a framework for the study of the mechanisms by which WNTs regulate diverse neuronal functions. Manipulation of the WNT pathways could provide new strategies for nerve regeneration and neuronal circuit modulation.     

Development of the limb neuromuscular system

Appendages, such as wings of a fly or limbs of a vertebrate, are excellent models to study the principles of patterning and morphogenesis. In the adult these structures are used for a variety of behaviors, including locomotion. Although support structures of the adult vertebrate limb are generated within the limb bud, its dynamic elements are derived from the somitic mesoderm and neural tube. Recent studies show that regional patterns set up in the mesenchyme-filled limb bud guide muscle precursors and developing motor axons to their proper location within the limb. Subsequent development of the neuromuscular system is regulated by cell surface interactions between pre-specified muscle fibers and motor axons.     

Wnt/Frizzled signaling controls C. elegans gastrulation by activating actomyosin contractility

BACKGROUND: Embryonic patterning mechanisms regulate the cytoskeletal machinery that drives morphogenesis, but there are few cases where links between patterning mechanisms and morphogenesis are well understood. We have used a combination of genetics, in vivo imaging, and cell manipulations to identify such links in C. elegans gastrulation. Gastrulation in C. elegans begins with the internalization of endodermal precursor cells in a process that depends on apical constriction of ingressing cells. RESULTS: We show that ingression of the endodermal precursor cells is regulated by pathways, including a Wnt-Frizzled signaling pathway, that specify endodermal cell fate. We find that Wnt signaling has a role in gastrulation in addition to its earlier roles in regulating endodermal cell fate and cell-cycle timing. In the absence of Wnt signaling, endodermal precursor cells polarize and enrich myosin II apically but fail to contract their apical surfaces. We show that a regulatory myosin light chain normally becomes phosphorylated on the apical side of ingressing cells at a conserved site that can lead to myosin-filament formation and contraction of actomyosin networks and that this phosphorylation depends on Wnt signaling. CONCLUSIONS: We conclude that Wnt signaling regulates C. elegans gastrulation through regulatory myosin light-chain phosphorylation, which results in the contraction of the apical surface of ingressing cells. These findings forge new links between cell-fate specification and morphogenesis, and they represent a novel mechanism by which Wnt signaling can regulate morphogenesis.     

FGF signaling regulates mesoderm cell fate specification and morphogenetic movement at the primitive streak.

Although FGF signaling plays an integral role in the migration and patterning of mesoderm at gastrulation, the mechanism and downstream targets of FGF activity have remained elusive. Here, we demonstrate that FGFR1 orchestrates the epithelial to mesenchymal transition and morphogenesis of mesoderm at the primitive streak by controlling Snail and E-cadherin expression. Furthermore, we show that FGFR1 functions in mesoderm cell fate specification by positively regulating Brachyury and Tbx6 expression. Finally, we provide evidence that the attenuation of Wnt3a signaling observed in Fgfr1 -/- embryos can be rescued by lowering E-cadherin levels. We propose that modulation of cytoplasmic beta-catenin levels, associated with FGF-induced downregulation of E-cadherin, provides a molecular link between FGF and Wnt signaling pathways at the streak.     

Emergent morphogenesis: Elastic mechanics of a self-deforming tissue

Multicellular organisms are generated by coordinated cell movements during morphogenesis. Convergent extension is a key tissue movement that organizes mesoderm, ectoderm, and endoderm in vertebrate embryos. The goals of researchers studying convergent extension, and morphogenesis in general, include understanding the molecular pathways that control cell identity, establish fields of cell types, and regulate cell behaviors. Cell identity, the size and boundaries of tissues, and the behaviors exhibited by those cells shape the developing embryo; however, there is a fundamental gap between understanding the molecular pathways that control processes within single cells and understanding how cells work together to assemble multicellular structures. Theoretical and experimental biomechanics of embryonic tissues are increasingly being used to bridge that gap. The efforts to map molecular pathways and the mechanical processes underlying morphogenesis are crucial to understanding: (1) the source of birth defects, (2) the formation of tumors and progression of cancer, and (3) basic principles of tissue engineering. In this paper, we first review the process of tissue convergent extension of the vertebrate axis and then review models used to study the self-organizing movements from a mechanical perspective. We conclude by presenting a relatively simple “wedge-model” that exhibits key emergent properties of convergent extension such as the coupling between tissue stiffness, cell intercalation forces, and tissue elongation forces.     

Wnt signal transduction pathways

The Wnt signaling pathway is an ancient and evolutionarily conserved pathway that regulates crucial aspects of cell fate determination, cell migration, cell polarity, neural patterning and organogenesis during embryonic development. The Wnts are secreted glycoproteins and comprise a large family of nineteen proteins in humans hinting to a daunting complexity of signaling regulation, function and biological output. To date major signaling branches downstream of the Fz receptor have been identified including a canonical or Wnt/β-catenin dependent pathway and the non-canonical or β-catenin-independent pathway which can be further divided into the Planar Cell Polarity and the Wnt/Ca²⁺ pathways, and these branches are being actively dissected at the molecular and biochemical levels. In this review, we will summarize the most recent advances in our understanding of these Wnt signaling pathways and the role of these pathways in regulating key events during embryonic patterning and morphogenesis.Key words: Wnt, frizzled, dishevelled, canonical, non-canonical, β-catenin, Planar Cell Polarity     

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.     

PI3K and Erk MAPK mediate ErbB signaling in Xenopus gastrulation

ErbB signaling regulates cell adhesion and movements during Xenopus gastrulation, but the downstream pathways involved have not been elucidated. In this study, we show that phosphatidylinositol-3 kinase (PI3K) and Erk mitogen-activated protein kinase (MAPK) mediate ErbB signaling to regulate gastrulation. Both PI3K and MAPK function sequentially in mesoderm specification and movements, and ErbB signaling is important only for the late phase activation of these pathways to control cell behaviors. Activation of either PI3K or Erk MAPK rescues gastrulation defects in ErbB4 morphant embryos, and restores convergent extension in the trunk mesoderm as well as coherent cell migration in the head mesoderm. The two signals preferentially regulate different aspects of cell behaviors, with PI3K more efficient in rescuing cell adhesion and spreading and MAPK more effective in stimulating the formation of filopodia. PI3K and MAPK also weakly activate each other, and together they modulate gastrulation movements. Our results reveal that PI3K and Erk MAPK, which have previously been considered as mesodermal inducing signals, also act downstream of ErbB signaling to participate in regulation of gastrulation morphogenesis.     

Cellular patterning of the vertebrate embryo

Recent studies show that cell dispersal is a widespread phenomenon in the development of early vertebrate embryos. These cell movements coincide with major decisions for the spatial organization of the embryo, and they parallel genetic patterning events. For example, in the central nervous system, cell dispersal is first mainly anterior–posterior and subsequently dorsal–ventral. Thus, genes expressed in signaling centers of the embryo probably control cell movements, tightly linking cellular and genetic patterning. Cell dispersal might be important for the correct positioning of cells and tissues involved in intercellular signaling. The emergence of cell dispersal at the onset of vertebrate evolution indicates a shift from early, lineage-based cellular patterning in small embryos to late, movement-based cellular patterning of polyclones in large embryos. The conservation of the same basic body plan by invertebrate and vertebrate chordates suggests that evolution of the embryonic period preceding the phylotypic stage was by intercalary co-option of basic cell activities present in the ancestral metazoan cell.