Expressions and localizations of Bax/Bcl-2 proteins during metamorphosis of Pelophylax ridibundus

Bcl-2 and Bax proteins are expressed in cells of the tails of Pelophylax ridibundus larvae. We investigated the levels of these proteins in tails undergoing apoptosis. Apoptotic cells were observed in the epidermis, muscle and notochord of tails of different lengths. The apoptotic cells in epidermis exhibited the typical features of apoptosis. Amorphous masses and irregularities in striated muscle tissue undergoing apoptosis and apoptotic remnants in the notochord also were observed. In general, Bax staining in the epidermis, subepidermal fibroblast layer, muscle and notochord cells increased, while Bcl-2 staining decreased as the tail regressed. Our results suggest that during tail regression due to metamorphosis, Bcl-2 and Bax proteins play key roles in the apoptosis of tail epidermis, subepidermal fibroblast layer, muscle and notochord cells.     

The determination of myogenic and cartilage cells in the early chick embryo and the modifying effect of retinoic acid

Summary The time of determination of cartilage and skeletal muscle was studied by making chimeric grafts or explants of small tissue pieces from several stages of early chick or quail embryos. Chondrogenesis was assessed by histology or with antibodies directed against type II collagen or cartilage proteoglycan, while myogenesis was detected immunohistochemically with antibodies directed against 3 different muscle markers, including muscle myosin. Grafts from Hensen's node, primitive streak and segmental plate of donor embryos of Stage 3–5 (Hamburger and Hamilton) were transplanted under the ectoderm in the extraembryonic area of Stage 12 host embryos. In addition, explants and mesodermal cells were cultured on glass in DMEM+F12 medium supplemented with 10% FCS. The results showed that determined myogenic cells could first be detected in Hensen's node and the primitive streak at Stage 3⁺–4 and that they developed from mesodermal cells located between the epiblast and hypoblast. Myogenic cells also appeared in grafted and explanted segmental plate with or without notochord from Stage 5 embryos. On the other hand, cartilage cells only formed in grafted and explanted segmental plate that also contained notochord. RA (1 ng/ml) could induce the formation of cartilage cells in the explanted primitive streak without Hensen's node or notochord taken from Stage 3–5 embryos and could also promote the differentiation of myogenic cells in primitive streak from Stage 3 embryo. Thus RA can substitute for Hensen's node or the notochord in the induction of cartilage cells and has some stimulatory effects on the differentiation of myogenic cells. Additional evidence indicates that the hypoblast might play an inductive role in the formation of the notochord which may subsequently promote the differentiation of cartilage cells. Offprint requests to: M. Solursh     

Nodal/Bozozok-independent induction of the dorsal organizer by zebrafish cell lines

Formation of the dorsal organizer (Spemann organizer) is an important process in early vertebrate development. In zebrafish, two molecular cascades—Bozozok/Dharma (Boz) and Nodal signaling—act in parallel to induce the dorsal organizer. However, the complete molecular mechanism regulating this event remains unclear. Here we report that zebrafish cell lines derived from various developmental stages can induce a secondary axis when they are implanted into the mid-blastula but not the early gastrula. The implanted cellsthemselves did not differentiate, but instead induced ectopic expression of dorsal organizer markers incells around the implanted cells and induced notochord formation in the secondary axis. These results indicate that cultured cell lines have the ability to induce a secondary axis through the initiation of dorsal organizer activity. However, ectopic expression of boz and sqt were not observed in cultured cells. In addition, implanted cell lines could induce the dorsal organizer even in maternal-zygotic one-eyed pinhead mutants, which are not responsive to Nodal signaling. Finally, the Nodal signaling pathway was not activatedfollowing implantation of cultured cells. Collectively, these data suggest that zebrafish cell lines induce the dorsal organizer independent of the boz and Nodal signaling pathways.     

The potentialities of transplanted early gastrula nuclei ofDrosophila melanogaster. Production of their imago descendants by germ-line transplantation

Summary Wild-type nuclei, taken out of cells from five regions of early gastrula embryos, were implanted singly into unfertilizedy w sn ³lz^50e eggs ofDrosophila melanogaster. The different types of nuclei initiated development with nearly equal frequencies of about 60%. 2.9% of the 1073 nuclear transfers developed as far as one of the three larval instars, and one reached the pupal stage.All individuals showed stage-specific patterns of defect. Most of these abnormalities were probably due to some inevitable damage caused by the implantation procedure such as disarrangement of the internal egg morphology and loss of peripheral egg substance. The proportions of individuals arrested at different embryonic and larval stages were similar for the five nuclear groups.Fertile imagos, descendants of all five types of donor nuclei, were produced via germ-line mosaics in two ways: (1) Pole cells of nuclear-transplant blastoderm stages were implanted into the pole cell region of host blastoderm eggs. (2) Gonads were taken from nuclear-transplant larvae and implanted into host larvae. In both cases gametes developed from the transplants as could be recognized from the genotypes of their progeny. By means of suitable crosses it was possible to get clones of flies whose large chromosomes were descended from the chromosomes of only one transplanted nucleus, that is, each clone was the descendant of one somatic nucleus. The data presented show that the nuclei remain omnipotent until the early gastrula stage.     

Absence of neural crest cells from the region surrounding implanted notochords in situ

Avian neural crest cells migrating along the trunk ventral pathway are distributed throughout the rostral half of the sclerotome with the exception of a neural crest cell-free space of approximately 85 microns width surrounding the notochord. To determine if this neural crest cell-free space results from the notochord inhibiting neural crest cell migration, a length of quail notochord was implanted lateral to the neural tube along the neural crest ventral migratory pathway of 2-day chicken embryos. The subsequent distribution of neural crest cells was analyzed in embryos fixed 2 days after grafting. When the donor notochord was isolated using collagenase, neural crest cells avoided the ectopic notochord and were absent from the area immediately surrounding the implant (mean distance of 43 microns). The neural crest cell-free space was significantly less when notochords were isolated using trypsin or chondroitinase digestion and was completely eliminated when notochords were fixed with paraformaldehyde or methanol prior to implantation. The implanted notochords did not appear to affect the overall number of neural crest cells, and therefore were unlikely to exert this effect by altering their viability. These results suggest that the notochord produces a substance that can inhibit neural crest cell migration and that this substance is trypsin and chondroitinase labile.     

Adult-type myogenesis of the frog <Emphasis Type="Italic">Xenopus laevis</Emphasis> specifically suppressed by notochord cells but promoted by spinal cord cells in vitro

Larval-to-adult myogenic conversion occurs in the dorsal muscle but not in the tail muscle during Xenopus laevis metamorphosis. To know the mechanism for tail-specific suppression of adult myogenesis, response character was compared between adult myogenic cells (Ad-cells) and larval tail myogenic cells (La-cells) to a Sonic hedgehog (Shh) inhibitor, notochord (Nc) cells, and spinal cord (SC) cells in vitro. Cyclopamine, an Shh inhibitor, suppressed the differentiation of cultured Ad (but not La) cells, suggesting the significance of Shh signaling in promoting adult myogenesis. To test the possibility that Shh-producing axial elements (notochord and spinal cord) regulate adult myogenesis, Ad-cells or La-cells were co-cultured with Nc or SC cells. The results showed that differentiation of Ad-cells were strongly inhibited by Nc cells but promoted by SC cells. If Ad-cells were “separately” co-cultured with Nc cells without direct cell–cell interactions, adult differentiation was not inhibited but rather promoted, suggesting that Nc cells have two roles, one is a short-range suppression and another is a long-range promotion for adult myogenesis. Immunohistochemical analysis showed both notochord and spinal cord express the N-terminal Shh fragment throughout metamorphosis. The “spinal cord-promotion” and long-range effect by Nc cells on adult myogenesis is thus involved in Shh signaling, while the signaling concerning the short-range “Nc suppression” will be determined by future studies. Interestingly, these effects, “Nc suppression” and “SC promotion” were not observed for La-cells. Situation where the spinal cord/notochord cross-sectional ratio is quite larger in tadpole trunk than in the tail seems to contribute to trunk-specific promotion and tail-specific suppression of adult myogenesis during Xenopus metamorphosis.     

Generation of knock‐in mice that express nuclear enhanced green fluorescent protein and tamoxifen‐inducible Cre recombinase in the notochord from Foxa2 and T loci

The node and the notochord are important embryonic signaling centers that control embryonic pattern formation. Notochord progenitor cells present in the node and later in the posterior end of the notochord move anteriorly to generate the notochord. To understand the dynamics of cell movement during notochord development and the molecular mechanisms controlling this event, analyses of cell movements using time‐lapse imaging and conditional manipulation of gene activities are required. To achieve this goal, we generated two knock‐in mouse lines that simultaneously express nuclear enhanced green fluorescent protein (EGFP) and tamoxifen‐inducible Cre, CreER^T2, from two notochord gene loci, Foxa2 and T (Brachury). In Foxa2^{nEGFP‐CreERT2/+} and T^{nEGFP‐CreERT2/+} embryos, nuclei of the Foxa2 or T‐expressing cells, which include the node, notochord, and endoderm (Foxa2) or wide range of posterior mesoderm (T), were labeled with EGFP at intensities that can be used for live imaging. Cre activity was also induced in cells expressing Foxa2 and T 1 day after tamoxifen administration. These mice are expected to be useful tools for analyzing the mechanisms of notochord development. genesis 51:210–218, 2013. © 2013 Wiley Periodicals, Inc.     

Wnt5 is required for notochord cell intercalation in the ascidian Halocynthia roretzi

Background information. In the embryos of various animals, the body elongates after gastrulation by morphogenetic movements involving convergent extension. The Wnt/PCP (planar cell polarity) pathway plays roles in this process, particularly mediolateral polarization and intercalation of the embryonic cells. In ascidians, several factors in this pathway, including Wnt5, have been identified and found to be involved in the intercalation process of notochord cells. Results. In the present study, the role of the Wnt5 genes, Hr‐Wnt5α (Halocynthia roretzi Wnt5α) and Hr‐Wnt5β, in convergent extension was investigated in the ascidian H. roretzi by injecting antisense oligonucleotides and mRNAs into single precursor blastomeres of various tissues, including notochord, at the 64‐cell stage. Hr‐Wnt5α is expressed in developing notochord and was essential for notochord morphogenesis. Precise quantitative control of its expression level was crucial for proper cell intercalation. Overexpression of Wnt5 proteins in notochord and other tissues that surround the notochord indicated that Wnt5α plays a role within the notochord, and is unlikely to be the source of polarizing cues arising outside the notochord. Detailed mosaic analysis of the behaviour of individual notochord cells overexpressing Wnt5α indicated that a Wnt5α‐manipulated cell does not affect the behaviour of neighbouring notochord cells, suggesting that Wnt5α works in a cell‐autonomous manner. This is further supported by comparison of the results of Wnt5α and Dsh (Dishevelled) knockdown experiments. In addition, our results suggest that the Wnt/PCP pathway is also involved in mediolateral intercalation of cells of the ventral row of the nerve cord (floor plate) and the endodermal strand. Conclusion. The present study highlights the role of the Wnt5α signal in notochord convergent extension movements in ascidian embryos. Our results raise the novel possibility that Wnt5α functions in a cell‐autonomous manner in activation of the Wnt/PCP pathway to polarize the protrusive activity that drives convergent extension.     

Did the notochord evolve from an ancient axial muscle? The axochord hypothesis

The origin of the notochord is one of the key remaining mysteries of our evolutionary ancestry. Here, we present a multi‐level comparison of the chordate notochord to the axochord, a paired axial muscle spanning the ventral midline of annelid worms and other invertebrates. At the cellular level, comparative molecular profiling in the marine annelids P. dumerilii and C. teleta reveals expression of similar, specific gene sets in presumptive axochordal and notochordal cells. These cells also occupy corresponding positions in a conserved anatomical topology and undergo similar morphogenetic movements. At the organ level, a detailed comparison of bilaterian musculatures reveals that most phyla form axochord‐like muscles, suggesting that such a muscle was already present in urbilaterian ancestors. Integrating comparative evidence at the cell and organ level, we propose that the notochord evolved by modification of a ventromedian muscle followed by the assembly of an axial complex supporting swimming in vertebrate ancestors.     

The effect of aging on the neural competence of the presumptive ectoderm and the effect of aged ectoderm on the differentiation of the trunk organizer inCynops pyrrhogaster

Summary The effect of aging on the neural competence of the presumptive ectoderm of the early gastrula, and the effect of aged ectoderm on the differentiation of the still uninvaginated dorsal blastoporal lip at the small yolk-plug stage — representing the trunk organizer — were examined by the sandwich method inCynops pyrrhogaster.The presumptive ectoderm to be used as reaction system was taken from 0 to 36 h exogastrulae obtained by operation at the early gastrula stage and combined with trunk organizer. In the 0 to 12 h explants typical trunktail structures were formed. With further aging of the presumptive ectoderm a decrease in frequency of spinal cord, notochord, and muscle and a simultaneous increase in frequency of mesenchyme and mesothelium were observed. In the 30 and 36 h explants neural competence had largely disappeared, the frequency of notochord and muscle become very low and their differentiation very poor, whereas the frequency of mesenchyme and mesothelium reached very high levels.We infer a reciprocal relationship between the induced spinal cord and the differentiation of notochord and muscle, as well as a transformation of notochordal material into mesenchyme and mesothelium under the influence of the aged ectoderm. The mode of action of the trunk organizer in normal development is discussed.     

Wnt signaling maintains the notochord fate for progenitor cells and supports the posterior extension of the notochord

The notochord develops from notochord progenitor cells (NPCs) and functions as a major signaling center to regulate trunk and tail development. NPCs are initially specified in the node by Wnt and Nodal signals at the gastrula stage. However, the underlying mechanism that maintains the NPCs throughout embryogenesis to contribute to the posterior extension of the notochord remains unclear. Here, we demonstrate that Wnt signaling in the NPCs is essential for posterior extension of the notochord. Genetic labeling revealed that the Noto-expressing cells in the ventral node contribute the NPCs that reside in the tail bud. Robust Wnt signaling in the NPCs was observed during posterior notochord extension. Genetic attenuation of the Wnt signal via notochord-specific β-catenin gene ablation resulted in posterior truncation of the notochord. In the NPCs of such mutant embryos, the expression of notochord-specific genes was down-regulated, and an endodermal marker, E-cadherin, was observed. No significant alteration of cell proliferation or apoptosis of the NPCs was detected. Taken together, our data indicate that the NPCs are derived from Noto-positive node cells, and are not fully committed to a notochordal fate. Sustained Wnt signaling is required to maintain the NPCs’ notochordal fate.     

The functional analysis of Type I postplasmic/PEM mRNAs in embryos of the ascidian Halocynthia roretzi

Maternal factors, such as a muscle determinant macho-1 mRNA that is localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, are crucial for embryonic axis formation and cell fate specification. Maternal mRNAs that show an identical posterior localization pattern to that of macho-1 in eggs and embryos are called Type I postplasmic/PEM mRNAs. We investigated the functions of five of the nine Type I mRNAs so far known in Halocynthia roretzi: Hr-Wnt-5, Hr-GLUT, Hr-PEM3, Hr-PEN1, and Hr-PEN2. Suppression of their functions with specific antisense morpholino oligonucleotides (MOs) had effects on the formation of various tissues: Hr-Wnt-5 on notochord, muscle, and mesenchyme, although zygotic function of Hr-Wnt-5 is responsible for notochord formation; Hr-GLUT on notochord, mesenchyme, and endoderm; and Hr-PEN2 on muscle, mesenchyme, and endoderm. On the other hand, Hr-PEM3 and Hr-PEN1 MOs seemed to have no effect. We conclude that the functions of at least some localized maternal Type I postplasmic/PEM mRNAs are necessary for early embryonic patterning in ascidians.     

Brachyury (T) Expression in Embryos of a Larvacean Urochordate, Oikopleura dioica, and the Ancestral Role of T

The Brachyury, or T, gene is required for notochord development in animals occupying all three chordate subphyla and probably also had this role in the last common ancestor of the chordate lineages. In two chordate subphyla (vertebrates and cephalochordates), T is also expressed during gastrulation in involuting endodermal and mesodermal cells, and in vertebrates at least, this expression domain is required for proper development. In the basally diverging chordate subphylum Urochordata, animals in the class Ascidiacea do not employ T during gastrulation in endodermal or nonaxial mesodermal cells, and it has been suggested that nonnotochordal roles for T were acquired in the cephalochordate–vertebrate lineage after it split with Urochordata. To test this hypothesis, we cloned T from Oikopleura dioica, a member of the urochordate class Appendicularia (or Larvacea), which diverged basally in the subphylum. Investigation of the expression pattern in developing Oikopleura embryos showed early expression in presumptive notochord precursor cells, in the notochord, and in parts of the developing gut and cells of the endodermal strand. We conclude that the ancestral role of T likely included expression in the developing gut and became necessary in chordates for construction of the notochord.     

Tube formation by complex cellular processes in Ciona intestinalis notochord

In the course of embryogenesis multicellular structures and organs are assembled from constituent cells. One structural component common to many organs is the tube, which consists most simply of a luminal space surrounded by a single layer of epithelial cells. The notochord of ascidian Ciona forms a tube consisting of only 40 cells, and serves as a hydrostatic “skeleton” essential for swimming. While the early processes of convergent extension in ascidian notochord development have been extensively studied, the later phases of development, which include lumen formation, have not been well characterized. Here we used molecular markers and confocal imaging to describe tubulogenesis in the developing Ciona notochord. We found that during tubulogenesis each notochord cell established de novo apical domains, and underwent a mesenchymal–epithelial transition to become an unusual epithelial cell with two opposing apical domains. Concomitantly, extracellular luminal matrix was produced and deposited between notochord cells. Subsequently, each notochord cell simultaneously executed two types of crawling movements bi-directionally along the anterior/posterior axis on the inner surface of notochordal sheath. Lamellipodia-like protrusions resulted in cell lengthening along the anterior/posterior axis, while the retraction of trailing edges of the same cell led to the merging of the two apical domains. As a result, the notochord cells acquired endothelial-like shape and formed the wall of the central lumen. Inhibition of actin polymerization prevented the cell movement and tube formation. Ciona notochord tube formation utilized an assortment of common and fundamental cellular processes including cell shape change, apical membrane biogenesis, cell/cell adhesion remodeling, dynamic cell crawling, and lumen matrix secretion.     

Pattern of Skeletal Muscle Differentiation in Fish: Molecular Biological Approaches

The initial stages of myogenesis going in myoblasts include the stages of induction, determination, and differentiation. The induction and determination of cells in the myotomes are controlled by morphogenetic signals from neighboring tissues of the notochord and neural tube manifested as expression of genes of Shh and Wnt families, respectively. In fish (at the example of danio), this signal is passed to somite cells neighboring the notochord; later the cells migrate to the embryo surface and differentiate into slow muscle fibers. Synthesis of the main contractile proteins, primarily the components of myosin molecule—heavy chain (MHC) and individual isoforms of light chains (MLC1, MLC2, and MLC3)—are encoded by different genes during different ontogenetic stages. The peptide maps obtained after -chymotrypsin digestion of MHCs from larvae, fast and slow skeletal muscle of loach are different, which points to differences in their primary structure. In addition, considerable differences were revealed in the structure of MLC isoforms at different ontogenetic stages. The definitive fast muscle contained three light chain types, MLC1, MLC2, and MLC3; slow muscle, MLC1 and MLC3; while the larval muscle fibers included a specific larval MLCL in addition to MLC3.